Conformationally constrained backbone cyclized peptide analogs

ABSTRACT

Novel backbone cyclized peptide analogs are formed by means of bridging groups attached via the alpha nitrogens of amino acid derivatives to provide novel non-peptidic linkages. Novel building units disclosed are N.sup.α (ω-functionalized) amino acids constructed to include a spacer and a terminal functional group. One or more of these N.sup.α (ω-functionalized) amino acids are incorporated into a peptide sequence, preferably during solid phase peptide synthesis. The reactive terminal functional groups are protected by specific protecting groups that can be selectively removed to effect either backbone-to-backbone or backbone-to-side chain cyclizations. The invention is exemplified by backbone cyclized bradykinin antagonists having biological activity. Further embodiments of the invention are somatostatin analogs having one or two ring structures involving backbone cyclization.

This application is a 371 of PCT/IB95/00453 filed Jun. 7, 1995.

FIELD OF THE INVENTION

The present invention relates to conformationally constrained N.sup.αbackbone-cyclized peptide analogs cyclized via novel non-peptidiclinkages, to novel N.sup.α,ω-functionalized amino acid building units,to processes for the preparation of these backbone cyclized peptides andbuilding units, to methods for using these peptide analogs and topharmaceutical compositions containing same.

BACKGROUND OF THE INVENTION Peptidomimetics

As a result of major advances in organic chemistry and in molecularbiology, many bioactive peptides can now be prepared in quantitiessufficient for pharmacological and clinical utilities. Thus in the lastfew years new methods have been established for the treatment andtherapy of illnesses in which peptides have been implicated. However,the use of peptides as drugs is limited by the following factors: a)their low metabolic stability towards proteolysis in thegastrointestinal tract and in serum; b) their poor absorption after oralingestion, in particular due to their relatively high molecular mass orthe lack of specific transport systems or both; c) their rapid excretionthrough the liver and kidneys; and d) their undesired side effects innon-target organ systems, since peptide receptors can be widelydistributed in an organism.

Moreover, with few exceptions, native peptides of small to medium size(less than 30-50 amino acids) exist unordered in dilute aqueous solutionin a multitude of conformations in dynamic equilibrium which may lead tolack of receptor selectivity, metabolic susceptibilities and hamperattempts to determine the biologically active conformation. If a peptidehas the biologically active conformation per se, i.e., receptor-boundconformation, then an increased affinity toward the receptor isexpected, since the decrease in entropy on binding is less than that onthe binding of a flexible peptide. It is therefore important to strivefor and develop ordered, uniform and biologically active peptides.

In recent years, intensive efforts have been made to developpeptidomimetics or peptide analogs that display more favorablepharmacological properties than their prototype native peptides. Thenative peptide itself, the pharmacological properties of which have beenoptimized, generally serves as a lead for the development of thesepeptidomimetics. However, a major problem in the development of suchagents is the discovery of the active region of a biologically activepeptide. For instance, frequently only a small number of amino acids(usually four to eight) are responsible for the recognition of a peptideligand by a receptor. Once this biologically active site is determined alead structure for development of peptidomimetic can be optimized, forexample by molecular modeling programs.

As used herein, a "peptidomimetic" is a compound that, as a ligand of areceptor, can imitate (agonist) or block (antagonist) the biologicaleffect of a peptide at the receptor level. The following factors shouldbe considered to achieve the best possible agonist peptidomimetic a)metabolic stability, b) good bioavailability, c) high receptor affinityand receptor selectivity, and d) minimal side effects.

From the pharmacological and medical viewpoint it is frequentlydesirable to not only imitate the effect of the peptide at the receptorlevel (agonism) but also to block the receptor when required(antagonism). The same pharmacological considerations for designing anagonist peptidomimetic mentioned above hold for designing peptideantagonists, but, in addition, their development in the absence of leadstructures is more difficult. Even today it is not unequivocally clearwhich factors are decisive for the agonistic effect and which are forthe antagonistic effect.

A generally applicable and successful method recently has been thedevelopment of conformationally restricted peptidomimetics that imitatethe receptor-bound conformation of the endogenous peptide ligands asclosely as possible (Rizo and Gierasch, Ann. Rev. Biochem., 61:387,1992). Investigations of these types of analogs show them to haveincreased resistance toward proteases, that is, an increase in metabolicstability, as well as increased selectivity and thereby fewer sideeffects (Veber and Friedinger, Trends Neurosci., p. 392, 1985).

Once these peptidomimetic compounds with rigid conformations areproduced, the most active structures are selected by studying theconformation-activity relationships. Such conformational constraints caninvolve short range (local) modifications of structure or long range(global) conformational restraints (for review see Giannis and Kolter,Angew. Chem. Int. Ed. Engl. 32:1244, 1993).

Conformationally Constrained Peptides

Bridging between two neighboring amino acids in a peptide leads to alocal conformational modification, the flexibility of which is limitedin comparison with that of regular dipeptides. Some possibilities forforming such bridges include incorporation of lactams and piperazinones.γ-Lactams and δ-lactams have been designed to some extent as "turnmimetics"; in several cases the incorporation of such structures intopeptides leads to biologically active compounds.

Global restrictions in the conformation of a peptide are possible bylimiting the flexibility of the peptide strand through cyclization(Hruby et al., Biochem. J., 268:249, 1990). Not only does cyclization ofbioactive peptides improve their metabolic stability and receptorselectivity, cyclization also imposes constraints that enhanceconformational homogeneity and facilitates conformational analysis. Thecommon modes of cyclization are the same found in naturally occurringcyclic peptides. These include side chain to side chain cyclization orside chain to end-group cyclization. For this purpose, amino acid sidechains that are not involved in receptor recognition are connectedtogether or to the peptide backbone. Another common cyclization is theend-to-end cyclization.

Three representative examples are compounds wherein partial structuresof each peptide are made into rings by linking two penicillamineresidues with a disulfide bridge (Mosberg et al., P.N.A.S. US, 80:5871,1983), by formation of an amide bond between a lysine and an aspartategroup (Charpentier et al., J. Med. Chem. 32:1184, 1989), or byconnecting two lysine groups with a succinate unit (Rodriguez et al.,Int. J. Pept. Protein Res. 35:441, 1990). These structures have beendisclosed in the literature in the case of a cyclic enkephalin analogwith selectivity for the δ-opiate receptor (Mosberg et al., ibid.); oras agonists to the cholecystokinin B receptor, found largely in thebrain (Charpentier et al., ibid., Rodriguez et al., ibid.).

The main limitations to these classical modes of cyclization are thatthey require substitution of amino acid side chains in order to achievecyclization.

Another conceptual approach to the conformational constraint of peptideswas introduced by Gilon, et al., (Biopolymers, 31:745, 1991) whoproposed backbone to backbone cyclization of peptides. The theoreticaladvantages of this strategy include the ability to effect cyclizationvia the carbons or nitrogens of the peptide backbone without interferingwith side chains that may be crucial for interaction with the specificreceptor of a given peptide. While the concept was envisaged as beingapplicable to any linear peptide of interest, in point of fact thelimiting factor in the proposed scheme was the availability of suitablebuilding units that must be used to replace the amino acids that are tobe linked via bridging groups. The actual reduction to practice of thisconcept of backbone cyclization was prevented by the inability to deviseany practical method of preparing building units of amino acids otherthan glycine (Gilon et al., J. Org. Chem., 587:5687, 1992). Whileanalogs of other amino acids were attempted the synthetic method usedwas unsuccessful or of such low yield as to preclude any generalapplicability.

In Gilon, EPO Application No. 564,739 A2; and J. Org. Chem., 57:5687,1992, two basic approaches to the synthesis of building units aredescribed. The first starts with the reaction of a diamine with ageneral α bromo acid. Selective protection of the ω amine and furtherelaborations of protecting groups provides a building unit, suitable forBoc chemistry peptide synthesis. The second approach starts withselective protection of a diamine and reaction of the product withchloroacetic acid to provide the protected glycine derivative, suitablefor Fmoc peptide synthesis.

Both examples deal with the reaction of a molecule of the general typeX--CH(R)--CO--OR' (wherein X represents a leaving group which, in theexamples given, is either Br or Cl) with an amine which replaces the X.The amine bears an alkylidene chain which is terminated by anotherfunctional group, amine in the examples described, which may or may notbe blocked by a protecting group.

In all cases the α nitrogen of the end product originates in themolecule which becomes the bridging chain for subsequent cyclization.This approach was chosen in order to take advantage of the highersusceptibility to nucleophilic displacement of a leaving group next to acarboxylic group.

In a molecule where R is different than hydrogen there is a hightendency to eliminate HX under basic conditions. This side reactionreduces the yield of Gilon's method to the point where it is impracticalfor production of building units based on amino acids other thanglycine. The diamine nitrogen is primary while the product contains asecondary nitrogen, which is a better nucleophile. So while the desiredreaction may be sluggish, and require the addition of catalysts, theproduct may be contaminated with double alkylation products. There is nomention of building units with end group chemistries other thannitrogen, so the only cyclization schemes possible are backbone to sidechain and backbone to C terminus.

Applications of Conformationally Constrained Peptides

Conformationally constrained peptides find many pharmacological uses.Somatostatin is a cyclic tetradecapeptide found both in the centralnervous system and in peripheral tissues. It was originally isolatedfrom mammalian hypothalamus and identified as an important inhibitor ofgrowth hormone secretion from the anterior pituitary. Its multiplebiological activities include inhibition of the secretion of glucagonand insulin from the pancreas, regulation of most gut hormones andregulation of the release of other neurotransmitters involved in motoractivity and cognitive processes throughout the central nervous system(for review see Lamberts, Endocrine Rev., 9:427, 1988).

Natural somatostatin (also known as Somatotropin Release InhibitingFactor, SRIF) of the following structure:

    H-Ala.sup.1 -Gly.sup.2 -Cys.sup.3 -Lys.sup.4 -Asn.sup.5 -Phe.sup.6 -Phe.sup.7 -Trp.sup.8 -Lys.sup.9 -Thr.sup.10 -Phe.sup.11 -Thr.sup.12 -Ser.sup.13 -Cys.sup.14 -OH

was first isolated by Guillemin and colleagues (Bruzeau et al. Science,179:78, 1973). In its natural form, it has limited use as a therapeuticagent since it exhibits two undesirable properties: poor bioavailabilityand short duration of action. For this reason, great efforts have beenmade during the last two decades to find somatostatin analogs that willhave superiority in either potency, biostability, duration of action orselectivity with regard to inhibition of the release of growth hormone,insulin or glucagon.

Structure-activity relation studies, spectroscopic techniques such ascircular dichroism and nuclear magnetic resonance, and molecularmodeling approaches reveal the following: the conformation of the cyclicpart of natural somatostatin is most likely to be an antiparallelβ-sheet; Phe⁶ and Phe¹¹ play an important role in stabilizing thepharmacophore conformation through hydrophobic interactions between thetwo aromatic rings; the four amino acids Phe⁷ -Trp⁸ -Lys⁹ -Thr¹⁰ whichare spread around the β-turn in the antiparallel β-sheet are essentialfor the pharmacophore; and (D)Trp⁸ is almost always preferable to(L)Trp⁸.

Nevertheless, a hexapeptide somatostatin analog containing these fouramino acids anchored by a disulfide bridge: ##STR1## is almost inactiveboth in vitro and in vivo, although it has the advantage of the covalentdisulfide bridge which replaces the Phe⁶ -Phe¹¹ hydrophobic interactionsin natural somatostatin.

Four main approaches have been attempted in order to increase theactivity of this hexapeptide somatostatin analog. (1) Replacing thedisulfide bridge by a cyclization which encourages a cis-amide bond, orby performing a second cyclization to the molecule yielding a bicyclicanalog. In both cases the resultant analog has a reduced number ofconformational degrees of freedom. (2) Replacing the original aminoacids in the sequence Phe⁷ -(D)Trp⁸ -Lys⁹ -Thr¹⁰ with more potent aminoacid analogs, such as replacing Phe⁷ with Tyr⁷ and Thr¹⁰ with Val¹⁰. (3)Incorporating additional structural elements from natural somatostatinwith the intention that these new elements will contribute to theinteraction with the receptor. (4) Eliminating one of the four aminoacids Phe⁷ -(D)Trp⁸ -Lys⁹ -Thr¹⁰ with the assumption that such analogswould be more selective.

The somatostatin analog, MK-678:

    cyclo(N-Me-Ala.sup.7 -Tyr.sup.7 -(D)Trp.sup.8 -Lys.sup.9 -Val.sup.10 -Phe)

is an example of a highly potent somatostatin analog designed using thefirst three approaches above (Veber, et al., Life Science, 34:371,1984). In this hexapeptide analog, a cisamide bond is located betweenN-Me-Ala and Phe¹¹, Tyr⁷ and Val¹⁰ replace Phe⁷ and Thr¹⁰ respectively,and Phe¹¹ is incorporated from natural somatostatin.

Another group of somatostatin analogs (U.S. Pat. Nos. 4,310,518 and4,235,886) includes octreotide: ##STR2## the only somatostatin analogcurrently available. It was developed using the third approach describedabove. Here, (D)Phe⁵ and the reduced C-terminal Thr¹² -CH₂ OH areassumed to occupy some of the conformational space available to thenatural Phe⁶ and Thr¹², respectively.

The compound TT2-32: ##STR3## is closely related to octreotide and is anexample of implementing the fourth approach described above. The lack ofThr¹⁰ is probably responsible for its high selectivity in terms ofantitumor activity.

These examples of highly potent somatostatin analogs indicate that thephenylalanines in positions 6 and 11 not only play an important role instabilizing the pharmacophore conformation but also have a functionalrole in the interaction with the receptor. It is still an open questionwhether one phenylalanine (either Phe⁶ or Phe¹¹) is sufficient for theinteraction with the receptor or whether both are needed.

It is now known that the somatostatin receptors constitute a family offive different receptor subtypes (Bell and Reisine, Trends Neurosci.,16, 34-38, 1993), which may be distinguished on the basis of theirtissue specificity and/or biological activity. Somatostatin analogsknown in the art may not provide sufficient selectivity or receptorsubtype selectivity, particularly as anti-neoplastic agents (Reubi andLaissue, TIPS, 16, 110-115, 1995).

Symptoms associated with metastatic carcinoid tumors (flushing anddiarrhea) and vasoactive intestinal peptide (VIP) secreting adenomas(watery diarrhea) are treated with somatostatin analogs. Somatostatinhas been also approved for the treatment of severe gastrointestinalhemorrhages. Somatostatin may also be useful in the palliative treatmentof other hormone-secreting tumors (e.g., pancreatic islet-cell tumorsand acromegaly) and hormone dependent tumors (e.g., chondrosarcoma andosteosarcoma) due to its anti-secretory activity.

Another important peptide, Bradykinin, is a naturally occurringnonapeptide,

    Arg.sup.1 -Pro.sup.2 -Pro.sup.3 -Gly.sup.4 -Phe.sup.5 -Ser.sup.6 -Pro.sup.7 -Phe.sup.8 -Arg.sup.9,

formed and released from precursors in the blood in response toinflammatory stimuli. Elevated levels of bradykinin also appear in otherbody fluids and tissues in pathological states such as asthma, septicshock and common cold. No clinical abnormalities have been associated sofar with bradykinin deficiency which indicates that bradykinin may notplay a critical role in normal physiology.

However, bradykinin mediates its physiological activities by binding toa specific receptive molecule called the bradykinin receptor. Two suchbradykinin receptors have been identified so far (these are called B1and B2 receptors). Subsequent to binding, the bradykinin signaltransduction pathway includes production of prostaglandins andleukotrienes as well as calcium activation. Through these mediators,bradykinin is involved in pain, inflammation, allergic reactions andhypotension. Therefore, a substance that can block the ability ofbradykinin to bind to its receptor, namely a bradykinin antagonist,should have a significant therapeutic value for one of the followingindications: asthma, inflammation, septic shock, pain, hypotension andallergy.

The analog used herein to exemplify backbone cyclization is:

    D-Arg.sup.0 -Arg-R.sup.1 -Hyp.sup.3 -Gly-Phe-R.sup.2 -D-Phe-Phe.sup.7 -Arg

(wherein, R¹ is Pro, R² is Ser in native bradykinin). The change ofproline at position 7 of native bradykinin to D-Phe confers antagonistactivity. This compound was described in Steranka, et al., P.N.A.S.U.S., 85:3245-3249, 1988 and is one of a plethora of candidate sequencesfor modification by the current technology, i.e. backbone cyclization.In this regard, it is worth noting the applications: WO 89/01781,EP-A-0370453 and EP-A-0334244 which disclose a wide range of candidatestructures. Antagonist peptides on which stability and/or tissueselectivity can be conferred by appropriate cyclization will be selectedfrom the many such known sequences.

According to the present invention a novel synthetic approach isdisclosed providing N.sup.α (ω(functionalized)alkylene) amino acidbuilding units that can be used to synthesize novel N.sup.α -backbonecyclized peptide analogs such as, but not limited to, novel somatostatinand bradykinin analogs. None of the above-mentioned references teachesor suggests N.sup.α -(ω(functionalized)alkylene) amino acids or thenovel N.sup.α -backbone cyclized peptide analogs of the presentinvention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide backbone cyclizedpeptide analogs that comprise peptide sequences which incorporate atleast two building units, each of which contains one nitrogen atom ofthe peptide backbone connected to a bridging group as described below.In the present invention, one or more pairs of the building units isjoined together to form a cyclic structure. Thus, according to oneaspect of the present invention, backbone cyclized peptide analogs areprovided that have the general Formula (I): ##STR4## wherein: a and beach independently designates an integer from 1 to 8 or zero; d, e, andf each independently designates an integer from 1 to 10; (AA) designatesan amino acid residue wherein the amino acid residues in each chain maybe the same or different; E represents a hydroxyl group, a carboxylprotecting group or an amino group, or CO--E can be reduced to CH₂ --OH;R and R' each designates an amino acid side-chain such as H, CH₃, etc.,optionally bound with a specific protecting group; and the linesindependently designate a bridging group of the Formula: (i)--X--M--Y--W--Z--; or (ii) --X--M--Z-- wherein: one line may be absent;M and W are independently selected from the group consisting ofdisulfide, amide, thioether, thioesters, imines, ethers and alkenes; andX, Y and Z are each independently selected from the group consisting ofalkylene, substituted alkylene, arylene, homo- or hetero-cycloalkyleneand substituted cycloalkylene.

In certain preferred embodiments, the CO--E group of Formula (I) isreduced to a CH₂ OH group.

Another embodiment of the present invention involves N-backbone to sidechain cyclized peptides of the general formula (II): ##STR5## whereinthe substituents are as defined above.

A preferred embodiment of the present invention involves the backbonecyclized peptide analog of Formulae I or II wherein the line designatesa bridging group of the Formula: --(CH₂)_(x) --M--(CH₂)_(y)--W--(CH₂)_(z) -- wherein M and W are independently selected from thegroup consisting of disulfide, amide, thioether, thioesters, imines,ethers and alkenes; x and z each independently designates an integerfrom 1 to 10, and y is zero or an integer of from 1 to 8, with theproviso that if y is zero, W is absent.

Further preferred are backbone cyclized peptide analogs of the Formula Ior II wherein R and R' are other than H, such as CH₃, (CH₃)₂ CH--,(CH₃)₂ CHCH₂ --, CH₃ CH₂ CH(CH₃)--, CH₃ S(CH₂)₂ --, HOCH₂ --, CH₃CH(OH)--, HSCH₂ --, NH₂ C(═O)CH₂ --, NH₂ C(═O)(CH₂)₂ --, NH₂ (CH₂)₃ --,HOC(═O)CH₂ --, HOC(═O)(CH₂)₂ --, NH₂ (CH₂)₄ --, C(NH₂)₂ NH(CH₂)₃ --,HO--phenyl--CH₂ --, benzyl, methylindole, and methylimidazole.

A more preferred embodiment of the present invention is directed tobackbone cyclization to stabilize the β-turn conformation of bradykininanalogs of the general Formula (III): ##STR6## wherein M is an amidebond, x and z are each independently an integer of 1 to 10, and K is Hor an acyl group.

Also more preferred are backbone cyclized peptide analogs of the presentinvention comprising bradykinin analogs of the general Formula (IVa):##STR7## wherein M is an amide bond, x and z are each independently aninteger of 1 to 10, K is H or an acyl group, and R⁶ is Gly or Ser; orthe general Formula (IVb): ##STR8## wherein x is an integer of 1 to 10;K is H or an acyl group; (R⁶) is selected from the group of D-Asp,L-Asp, D-Glu and L-Glu; and z is according to the amino acid specified:1 in case of D and L-Asp, and 2 in the case of D and L Glu.

Further more preferred backbone cyclized peptide analogs according tothe present invention having bradykinin antagonist activity have theFormula (V): ##STR9## wherein M is an amide bond, x and z are eachindependently an integer of 1 to 10, and K is H or an acyl group.

Specifically preferred backbone cyclized peptide analogs of the presentinvention are:

1) Ada-(D)Arg-Arg-cyclo(N.sup.α(1-(6-aminohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg--OH;

2) H-D-Arg-Arg-cyclo(N.sup.α (1-(4-propanoyl))Gly-Hyp-Phe-N.sup.α(3-amido-propylene)Gly)-Ser-D-Phe-Phe-Arg--OH; and

3) H-D-Arg-Arg-cyclo(N.sup.α (4-propanoyl)Gly-Hyp-Phe-N.sup.α(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg--OH.

Another preferred aspect of the present invention is directed tobackbone cyclization to generate novel somatostatin analogs linkedbetween positions 6 and 11, leaving the phenylalanine side chainsuntouched. This conformational stabilization is much more rigid than thePhe⁶, Phe¹¹ hydrophobic interaction in natural somatostatin and is morestable to reduction/oxidation reactions than the Cys--Cys disulfidebridge. In other words, for the first time a stable covalent bridge canbe achieved while either one or both of the original Phe⁶ and Phe¹¹ areretained.

Moreover, backbone cyclizations can also be used to anchor the β-turn,not only in positions 6 and 11 but also inside the active reaction ofPhe⁷ -(D)Trp⁸ -Lys9-Thr¹⁰, yielding either a monocyclic analog with apreferable conformation or a very rigid bicyclic analog. Here again, theside chains of the pharmacologically active amino acids remain untouchedand the only change is in limiting the conformational space.

As used herein and in the claims in the following more preferredbackbone cyclized peptide analogs, the superscript numbers following theamino acids refer to their position numbers in the native Somatostatin.

A more preferred backbone cyclized peptide novel analog is the Formula(XIVa): ##STR10## with a most preferred analog being the Formula (XIVb):##STR11##

wherein m and n are 1, 2 or 3; X is CH₂ OH or CONH₂ ; R⁵ is absent or isGly, (D)- or (L)-Ala, Phe, Nal and β-Asp(Ind); R⁶ and R¹¹ areindependently Gly or (D)- or (L)-Phe; R⁷ is Phe or Tyr; R¹⁰ is absent oris Gly, Abu, Thr or Val; R¹² is absent or is Thr or Nal, and Y² isselected from the group consisting of amide, disulfide, thioether,imines, ethers and alkenes. In these monocyclic somatostatin analogs, abackbone cyclization replaces the Cys⁶ -Cys¹¹ disulfide bridge, leavingthe phenylalanine side chains as in the natural somatostatin. Still morepreferred is the analog wherein Phe⁷ is replaced with Tyr⁷ and Thr¹⁰ isreplaced with Val¹⁰.

Other more preferred monocyclic analogs that anchor the molecule inpositions inside the active region rather than in positions 6 and 11 areformulae XV (a and b) and XVI (a-c): ##STR12## wherein i and j areindependently 1, 2 or 3; X is CH₂ OH or CONH₂ ; R⁵ is absent or is (D)-or (L)-Phe, Nal, or β-Asp(Ind); R⁶ is (D) or (L)-Phe; R¹⁰ is absent oris Gly, Abu or Thr; and is (D)- or (L)-Phe; R¹² is absent or is Thr orNal, and Y¹ is selected from the group consisting of amide, disulfide,thioether, imines, ethers and alkenes.

Still other more preferred analogs incorporate backbone cyclization inpositions 6 and 11 as in Formula XIV, together with the backbonecyclizations as in Formula XV and XVI, yielding rigid bicyclic analogsof the Formulae XVII (a and b) and XVIII (a and b): ##STR13##

wherein i, j, m and n are independently 1, 2 or 3; X is CH₂ OH or NH₂ ;R⁵ is absent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ and R¹¹ areindependently Gly or (D)- or (L)-Phe; R¹⁰ is absent or is Gly, Abu, Valor Thr; R¹² is absent or is Thr or Nal; and Y¹ and Y² are independentlyselected from the group consisting of amide, disulfide, thioether,imines, ethers and alkenes.

Other more preferred bicyclic analogs differ from Formulae XVII andXVIII by the replacement of the amino acids at positions 6 and 11 bycysteines which form a disulfide bond, leaving only one backbonecyclization in the Formulae XIX (a and b) and XX (a and b): ##STR14##

wherein i and j are independently 1, 2 or 3; X is CH₂ OH or NH₂ ; R⁵ isabsent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ and R¹¹ areindependently Gly or Phe; R¹⁰ is absent or is Gly, Abu or Thr; R¹² isabsent or is Thr or Nal; and Y¹ is selected from the group consisting ofamide, disulfide, thioether, imines, ethers and alkenes.

Another aspect of the present invention is a method for the preparationof cyclic peptides of the general Formula (I): ##STR15##

wherein: a and b each independently designates an integer from 1 to 8 orzero; d, e, and f each independently designates. an integer from 1 to10; (AA) designates an amino acid residue wherein the amino acidresidues in each chain may be the same or different; E represents ahydroxyl group, a carboxyl protecting group or an amino group, or CO--Ecan be reduced to CH₂ --OH; R and R' each designates an amino acidside-chain optionally bound with a specific protecting group; and thelines designate a bridging group of the Formula:

    --X--M--Y--W--Z--;                                         (i)

or

    --X--M--Z--                                                (ii)

wherein: one line may be absent; M and W are independently selected fromthe group consisting of disulfide, amide, thioether, thioesters, imines,ethers and alkenes; and X, Y and Z are each independently selected fromthe group consisting of alkylene, substituted alkylene, arylene, homo-orhetero-cycloalkylene and substituted cycloalkylene. This methodcomprises the steps of incorporating at least one N.sup.α-ω-functionalized derivative of amino acids of Formula (VI): ##STR16##wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R' is an amino acid side chain, optionally bound with aspecific protecting group; B is a protecting group selected from thegroup consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls;and G is a functional group selected from the group consisting ofamines, thiols, alcohols, carboxylic acids and esters, aldehydes,alcohols and alkyl halides; and A is a specific protecting group of G;into a peptide sequence and subsequently selectively cyclizing thefunctional group with one of the side chains of the amino acids in saidpeptide sequence or with another ω-functionalized amino acid derivative.

A further object of the present invention is directed to building unitsknown as a N.sup.α -ω-functionalized derivatives of the general Formula(VI) of amino acids which are prerequisites for the cyclization process:##STR17##

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R is the side chain of an amino acid, optionally boundwith a specific protecting group; B is a protecting group selected fromthe group consisting of alkyloxy, substituted alkyloxy, or aryloxycarbonyls; and G is a functional group selected from the groupconsisting of amines, thiols, alcohols, carboxylic acids and esters,aldehydes and alkyl halides; and A is a protecting group thereof.

Preferred building units are the ω-functionalized amino acid derivativeswherein X is alkylene; G is a thiol group, an amine group or a carboxylgroup; R is phenyl, methyl or isobutyl; with the proviso that when G isan amine group, R is other than H.

Further preferred are ω-functionalized amino acid derivatives wherein Ris protected with a specific protecting group.

More preferred are ω-functionalized amino acid derivatives of theFormulae: ##STR18## wherein X, R, A and B are as defined above.

Specifically preferred ω-functionalized amino acid derivatives includethe following:

1) N.sup.α -(Fmoc)(3-Boc-amino propylene)-(S)Phenylalanine;

2) N.sup.α -(Fmoc)(3-Boc-amino propylene)-(R)Phenylalanine;

3) N.sup.α -(Fmoc)(4-Boc-amino butylene)-(S)Phenylalanine;

4) N.sup.α -(Fmoc)(3-Boc-amino propylene)-(S)Alanine;

5) N.sup.α -(Fmoc)(6-Boc-amino hexylene)-(S)Alanine;

6) N.sup.α -(Fmoc)(3-Boc-amino propylene)-(R)Alanine;

7) N.sup.α -(2-(benzylthio)ethylene)glycine ethyl ester;

8) N.sup.α -(2-(benzylthio)ethylene)(S)leucine methyl ester;

9) N.sup.α -(3-(benzylthio)propylene)(S)leucine methyl ester:

10) Boc-N.sup.α -(2-(benzylthio)ethylene)glycine;

11) Boc-N.sup.α -(2-(benzylthio)ethylene)(S)phenylalanine;

12) Boc-N.sup.α -(3-(benzylthio)propylene)(S)phenylalanine;

13) Boc-L-phenylalanyl-N.sup.α -(2-(benzylthio)ethylene)glycine-ethylester;

14) Boc-L-phenylalanyl-N.sup.α-(2-(benzylthio)ethylene)-(S)phenylalanine methyl ester;

15) N.sup.α (Fmoc)-(2-t-butyl carboxy ethylene)glycine;

16) N.sup.α (Fmoc)-(3-t-butyl carboxy propylene)glycine;

17) N.sup.α (Fmoc)(2-t-butyl carboxy ethylene)(S)phenylalanine;

18) N.sup.α (Fmoc)(2-Boc amino ethylene)glycine;

19) N.sup.α (Fmoc)(3-Boc amino propylene)glycine;

20) N.sup.α (Fmoc)(4-Boc amino butylene)glycine; and

21) N.sup.α (Fmoc)(6-Boc amino hexylene)glycine.

Novel, practical, generally applicable processes for the preparation ofthese N.sup.α -ω-functionalized derivatives of amino acids are a furtheraspect of this invention.

As such, an object of this invention is a method of making anω-functionalized amino acid derivative of the general Formula: ##STR19##

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R is the side chain of an amino acid, such as H, CH₃,etc.; A and B are protecting groups selected from the group consistingof alkyloxy, substituted alkyloxy, or aryloxy carbonyls;

comprising the steps of:

i) reacting a diamine compound of the general Formula: ##STR20##

wherein A, B and X are as defined above,

with a triflate of Formula CF₃ SO₂ --O--CH(R)--CO--E wherein E is acarboxyl protecting group and R is as defined above; to yield a compoundof Formula: ##STR21##

wherein A, B, E, R and X are as defined above

ii) and deprotecting the carboxyl to yield an N.sup.α ω-functionalizedamino acid derivative, wherein the ω-functional group is an amine.

A further object of this invention is a method of making anω-functionalized amino acid derivative of the general Formula: ##STR22##

where B is a protecting group selected from the group of substitutedalkyloxy, substituted alkyloxy, or aryloxy carbonyls; R is the sidechain of an amino acid, such as H, CH₃, etc.; X is a spacer groupselected from the group of alkylene, substituted alkylene, arylene,cycloalkylene or substituted cycloalkylene; and A is a protecting groupselected from the group of alkyl or substituted alkyl, thio ethers oraryl or substituted aryl thio ethers;

comprising the steps of:

i) reacting a compound of the general Formula B--NH--X--S--A with atriflate of the general Formula CF₃ SO₂ --O--CH(R)--CO--E wherein E is acarboxyl protecting group and A, X and R are as defined above, to give acompound of the Formula: ##STR23## ii) selectively removing theprotecting group E, and iii) protecting the free amino group to yield anN.sup.α (ω-functionalized) amino acid derivative, wherein theω-functional group is a thiol.

A further object of this invention is a method of making anω-functionalized amino acid derivative of the general Formula: ##STR24##

where B is a protecting group selected from the group of alkyloxy,substituted alkyloxy, or aryloxy carbonyls; R is the side chain of anamino acid, such as H, CH₃, etc.; X is a spacer group selected from thegroup of alkylene, substituted alkylene, arylene, cycloalkylene orsubstituted cycloalkylene; and A is a protecting group selected from thegroup of alkyl or substituted alkyl, esters, or thio esters orsubstituted aryl esters or thio esters;

comprising the steps of:

i) reacting a compound of the general Formula B--NH--X--CO--A with atriflate of the general Formula CF₃ SO₂ --O--CH(R)--CO--E wherein E is acarboxyl protecting group and A, B, X and R are as defined above, togive a compound of Formula: ##STR25## ii) and selectively removingprotecting group E, to yield an N.sup.α (ω-functionalized) amino acidderivative, wherein the ω-functional group is a carboxyl.

A further aspect of this invention is to provide methods for thepreparation of novel backbone cyclic peptides, comprising the steps ofincorporating at least one N.sup.α -ω-functionalized derivatives ofamino acids into a peptide sequence and subsequently selectivelycyclizing the functional group with one of the side chains of the aminoacids in said peptide sequence, or with another ω-functionalized aminoacid derivative.

Backbone cyclized analogs of the present invention may be used aspharmaceutical compositions and for methods for the treatment ofdisorders including: acute asthma, septic shock, brain trauma and othertraumatic injury, post-surgical pain, all types of inflammation,cancers, endocrine disorders and gastrointestinal disorders.

Therefore, further objects of the present invention are directed topharmaceutical compositions comprising pharmacologically active backbonecyclized peptide agonists and antagonists prepared according to themethods disclosed herein and a pharmaceutically acceptable carrier ordiluent; and methods for the treatment of inflammation, septic shock,cancer or endocrine disorders and gastrointestinal disorders therewith.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing in vitro biostability of somatostatin andthree analogs thereof in human serum. The graph depicts the percentageof undegraded molecules for each of the compounds initially and aftervarious periods of time.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

All abbreviations used are in accordance with the IUPACIUBrecommendations on Biochemical Nomenclature (J. Biol. Chem.,247:977-983, 1972) and later supplements.

As used herein and in the claims, the phrase "an amino acid side chain"refers to the distinguishing substituent attached to the α-carbon of anamino acid; such distinguishing groups are well known to those skilledin the art. For instance, for the amino acid glycine, the R group is H;for the amino acid alanine, R is, CH₃, and so on. Other typical sidechains of amino acids include the groups: (CH₃)₂ CH--, (CH₃)₂ CHCH₂ --,CH₃ CH₂ CH(CH₃)--, CH₃ S(CH₂)₂ --, HOCH₂ --, CH₃ CH(OH)--, HSCH₂ --, NH₂C(═O)CH₂ --, NH₂ C(═O)(CH₂)₂ --, NH₂ (CH₂)₃ --, HOC(═O)CH₂ --,HOC(═O)(CH₂)₂ --, NH₂ (CH₂)₄ --, C(NH₂)₂ NH(CH₂)₃ --, HO--phenyl--CH₂--, benzyl, methylindole, and methylimidazole.

As used herein and in the claims, the letters "(AA)" and the term "aminoacid" are intended to include common natural or synthetic amino acids,and common derivatives thereof, known to those skilled in the art,including but not limited to the following. Typical amino-acid symbolsdenote the L configuration unless otherwise indicated by D appearingbefore the symbol.

    ______________________________________    Abbreviated Designation                      Amino Acids    ______________________________________    Abu               α-Amino butyric acid    Ala               L-Alanine    Arg               L-Arginine    Asn               L-Asparagine    Asp               L-Aspartic acid    βAsp(Ind)    β-Indolinyl aspartic acid    Cys               L-Cysteine    Glu               L-Glutamic acid    Gln               L-Glutamine    Gly               Glycine    His               L-Histidine    Hyp               trans-4-L-Hydroxy Proline    Ile               L-Isoleucine    Leu               L-Leucine    Lys               L-Lysine    Met               L-Methionine    Nal               β-Naphthyl alanine    Orn               Ornithine    Phe               L-Phenylalanine    Pro               L-Proline    Ser               L-Serine    Thr               L-Threonine    Trp               L-Tryptophane    Tyr               L-Tyrosine    Val               L-Valine    ______________________________________

Typical protecting groups, coupling agents, reagents and solvents suchas but not limited to those listed below have the followingabbreviations as used herein and in the claims. One skill in the artwould understand that the compounds listed within each group may be usedinterchangeably; for instance, a compound listed under "reagents andsolvents" may be used as a protecting group, and so on. Further, oneskill in the art would know other possible protecting groups, couplingagents and reagents/solvents; these are intended to be within the scopeof this invention.

    ______________________________________    Abbreviated Designation    ______________________________________                    Protecting Groups    Ada             Adamantane acetyl    Alloc           Allyloxycarbonyl    Allyl           Allyl ester    Boc             tert-butyloxycarbonyl    Bzl             Benzyl    Fmoc            Fluorenylmethyloxycarbonyl    OBzl            Benzyl ester    OEt             Ethyl ester    OMe             Methyl ester    Tos (Tosyl)     p-Toluenesulfonyl    Trt             Triphenylmethyl    Z               Benzyloxycarbonyl                    Coupling Agents    BOP             Benzotriazol-1-yloxytris-                    (dimethyl-amino)phosphonium                    hexafluorophosphate    DIC             Diisopropylcarbodiimide    HBTU            2-(1H-Benzotriazol-1-yl)-                    1,1,3,3-tetramethyluronium                    hexafluorophosphate    PyBrOP          Bromotripyrrolidinophosphonium                    hexafluorophosphate    PyBOP           Benzotriazol-1-yl-oxy-tris-                    pyrrolidino-phosphonium                    hexafluorophosphate    TBTU            O-(1,2-dihydro-2-oxo-1-pyridyl)-                    N,N,N',N'-tetramethyluronium                    tetrafluoroborate                    Reagents                    and Solvents    ACN             Acetonitrile    AcOH            Acetic acid    Ac.sub.2 O      Acetic acid anhydride    AdacOH          Adamantane acetic acid    Alloc-Cl        Allyloxycarbonyl chloride    Boc.sub.2 O     Di-tert butyl dicarbonate    DMA             Dimethylacetamide    DMF             N,N-dimethylformamide    DIEA            Diisopropylethylamine    Et.sub.3 N      Triethylamine    EtOAc           Ethyl acetate    FmocOSu         9-fluorenylmethyloxy carbonyl                    N-hydroxysuccinimide ester    HOBT            1-Hydroxybenzotriazole    HF              Hydrofluoric acid    MeOH            Methanol    Mes (Mesyl)     Methanesulfonyl    NMP             1-methyl-2-pyrrolidinone    nin.            Ninhydrin    i-PrOH          Iso-propanol    Pip             Piperidine    PP              4-pyrrolidinopyridine    Pyr             Pyridine    SRIF            Somatotropin release inhibiting                    factor    SST             Somatostatin    SSTR            Somatostatin receptor    TEA             Triethylamine    TFA             Trifluoroacetic acid    THF             Tetrahydrofuran    Triflate (Trf)  Trifluoromethanesulfonyl    Trf.sub.2 O     Trifluoromethanesulfonic acid                    anhydride    ______________________________________

The compounds herein described may have asymmetric centers. All chiral,diastereomeric, and racemic forms are included in the present invention.Many geometric isomers of olefins and the like can also be present inthe compounds described herein, and all such stable isomers arecontemplated in the present invention.

By "stable compound" or "stable structure" is meant herein a compoundthat is sufficiently robust to survive isolation to a useful degree ofpurity from a reaction mixture, and Formulation into an efficacioustherapeutic agent.

As used herein and in the claims, "alkyl" or "alkylenyl" is intended toinclude both branched and straight-chain saturated aliphatic hydrocarbongroups having one to ten carbon atoms; "alkenyl" is intended to includehydrocarbon chains of either a straight or branched configuration andone or more unsaturated carbon--carbon bonds which may occur in anystable point along the chain, such as ethenyl, propenyl, and the like;and "alkynyl" is intended to include hydrocarbon chains of either astraight or branched configuration and one or more triple carbon-carbonbonds which may occur in any stable point along the chain, such asethynyl, propynyl, and the like.

As used herein and in the claims, "aryl" is intended to mean any stable5- to 7-membered monocyclic or bicyclic or 7- to 14-membered bicyclic ortricyclic carbon ring, any of which may be saturated, partiallyunsaturated or aromatic, for example, phenyl, naphthyl, indanyl, ortetrahydronaphthyl tetralin, etc.

As used herein and in the claims, "alkyl halide" is intended to includeboth branched and straight-chain saturated aliphatic hydrocarbon groupshaving one to ten carbon atoms, wherein 1 to 3 hydrogen atoms have beenreplaced by a halogen atom such as Cl, F, Br, and I.

As used herein and in the claims, the term "heterocyclic" is intended tomean any stable 5- to 7-membered monocyclic or bicyclic or 7- to10-membered bicyclic heterocyclic ring, which is either saturated orunsaturated, and which consists of carbon atoms and from 1 to 3heteroatoms selected from the group consisting of N, O and S and whereinthe nitrogen and sulfur atoms may optionally be oxidized, and thenitrogen atom optionally be quaternized, and including any bicyclicgroup in which any of the above-defined heterocyclic rings is fused to abenzene ring. The heterocyclic ring may be attached to its pendant groupat any heteroatom or carbon atom which results in a stable structure.The heterocyclic rings described herein may be substituted on carbon oron a nitrogen atom if the resulting compound is stable. Examples of suchheterocycles include, but are not limited to pyridyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, benzothiophenyl, indolyl, indolenyl, quinolinyl,piperidonyl, pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oroctahydroisoquinolinyl and the like.

As used herein and in the claims, the phrase "therapeutically effectiveamount" means that amount of novel backbone cyclized peptide analog orcomposition comprising same to administer to a host to achieve thedesired results for the indications described herein, such as but notlimited of inflammation, septic shock, cancer, endocrine disorders andgastrointestinal disorders.

The term, "substituted" as used herein and in the claims, means that anyone or more hydrogen atoms on the designated atom is replaced with aselection from the indicated group, provided that the designated atom'snormal valency is not exceeded, and that the substitution results in astable compound.

When any variable (for example R, x, z, etc.) occurs more than one timein any constituent or in Formulae (I to XX) or any other Formula herein,its definition on each occurrence is independent of its definition atevery other occurrence. Also, combinations of substituents and/orvariables are permissible only if such combinations result in stablecompounds.

Synthetic Approach

According to the present invention peptide analogs are cyclized viabridging groups attached to the alpha nitrogens of amino acids thatpermit novel non-peptidic linkages. In general, the procedures utilizedto construct such peptide analogs from their building units rely on theknown principles of peptide synthesis; most conveniently, the procedurescan be performed according to the known principles of solid phasepeptide synthesis. The innovation requires replacement of one or more ofthe amino acids in a peptide sequence by novel building units of thegeneral Formula: ##STR26## wherein R is the side chain of an amino acid,X is a spacer group and G is the functional end group by means of whichcyclization will be effected. The side chain R is the side chain of anynatural or synthetic amino acid that is selected to be incorporated intothe peptide sequence of choice. X is a spacer group that is selected toprovide a greater or lesser degree of flexibility in order to achievethe appropriate conformational constraints of the peptide analog. Suchspacer groups include alkylene chains, substituted, branched andunsaturated alkylenes, arylenes, cycloalkylenes, unsaturated andsubstituted cycloakylenes. Furthermore, X and R can be combined to forma heterocyclic structure.

A preferred embodiment of the present invention utilizes alkylene chainscontaining from two to ten carbon atoms.

The terminal (ω) functional groups to be used for cyclization of thepeptide analog include but are not limited to:

a. Amines, for reaction with electrophiles such as activated carboxylgroups, aldehydes and ketones (with or without subsequent reduction),and alkyl or substituted alkyl halides.

b. Alcohols, for reaction with electrophiles such as activated carboxylgroups.

c. Thiols, for the formation of disulfide bonds and reaction withelectrophiles such as activated carboxyl groups, and alkyl orsubstituted alkyl halides.

d. 1,2 and 1,3 Diols, for the formation of acetals and ketals.

e. Alkynes or Substituted Alkynes, for reaction with nucleophiles suchas amines, thiols or carbanions; free radicals; electrophiles such asaldehydes and ketones, and alkyl or substituted alkyl halides; ororganometallic complexes.

f. Carboxylic Acids and Esters, for reaction with nucleophiles (with orwithout prior activation), such as amines, alcohols, and thiols.

g. Alkyl or Substituted Alkyl Halides or Esters, for reaction withnucleophiles such as amines, alcohols, thiols, and carbanions (fromactive methylene groups such as acetoacetates or malonates); andformation of free radicals for subsequent reaction with alkenes orsubstituted alkenes, and alkynes or substituted alkynes.

h. Alkyl or Aryl Aldehydes and Ketones for reaction with nucleophilessuch as amines (with or without subsequent reduction), carbanions (fromactive methylene groups such as acetoacetates or malonates), diols (forthe formation of acetals and ketals).

i. Alkenes or Substituted Alkenes, for reaction with nucleophiles suchas amines, thiols, carbanions, free radicals, or organometalliccomplexes.

j. Active Methylene Groups, such as malonate esters, acetoacetateesters, and others for reaction with electrophiles such as aldehydes andketones, alkyl or substituted alkyl halides.

It will be appreciated that during synthesis of the peptide thesereactive end groups, as well as any reactive side chains, must beprotected by suitable protecting groups. Suitable protecting groups foramines ate alkyloxy, substituted alkyloxy, and aryloxy carbonylsincluding, but not limited to, tert butyloxycarbonyl (Boc),Fluorenylmethyloxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc) andBenzyloxycarbonyl (Z).

Carboxylic end groups for cyclizations may be protected as their alkylor substituted alkyl esters or thio esters or aryl or substituted arylesters or thio esters. Examples include but are not limited to tertiarybutyl ester, allyl ester, benzyl ester, 2-(trimethylsilyl)ethyl esterand 9-methyl fluorenyl.

Thiol groups for cyclizations may be protected as their alkyl orsubstituted alkyl thio ethers or disulfides or aryl or substituted arylthio ethers or disulfides. Examples of such groups include but are notlimited to tertiary butyl, trityl(triphenylmethyl), benzyl,2-(trimethylsilyl)ethyl, pixyl(9-phenylxanthen-9-yl), acetamidomethyl,carboxy-methyl, 2-thio-4-nitropyridyl.

It will further be appreciated by the artisan that the various reactivemoieties will be protected by different protecting groups to allow theirselective removal. Thus, a particular amino acid will be coupled to itsneighbor in the peptide sequence when the N.sup.α is protected by, forinstance, protecting group A. If an amine is to be used as an end groupfor cyclization in the reaction scheme the N.sup.ω will be protected byprotecting group B, or an ε amino group of any lysine in the sequencewill be protected by protecting group C, and so on.

The coupling of the amino acids to one another is performed as a seriesof reactions as is known in the art of peptide synthesis. Novel buildingunits of the invention, namely the N.sup.α -ω functionalized amino acidderivatives are incorporated into the peptide sequence to replace one ormore of the amino acids. If only one such N.sup.α -ω functionalizedamino acid derivative is selected, it will be cyclized to a side chainof another amino acid in the sequence. For instance: (a) an N.sup.α-(ω-amino alkylene) amino acid can be linked to the carboxyl group of anaspartic or glutamic acid residue; (b) an N.sup.α -(ω-carboxylicalkylene) amino acid can be linked to the ε-amino group of a lysineresidue; (c) an N.sup.α -(ω-thio alkylene) amino acid can be linked tothe thiol group of a cysteine residue; and so on. A more preferredembodiment of the invention incorporates two such N.sup.α-ω-functionalized amino acid derivatives which may be linked to oneanother to form N-backbone to N-backbone cyclic peptide analogs. Threeor more such building units can be incorporated into a peptide sequenceto create bi-cyclic peptide analogs as will be elaborated below. Thus,peptide analogs can be constructed with two or more cyclizations,including N-backbone to N-backbone, as well as backbone to side-chain orany other peptide cyclization.

As stated above, the procedures utilized to construct peptide analogs ofthe present invention from novel building units generally rely on theknown principles of peptide synthesis. However, it will be appreciatedthat accommodation of the procedures to the bulkier building units ofthe present invention may be required. Coupling of the amino acids insolid phase peptide chemistry can be achieved by means of a couplingagent such as but not limited to dicyclohexycarbodiimide (DCC),bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl),benzotriazolyl-N-oxytrisdimethyl-aminophosphonium hexafluoro phosphate(BOP), 1-oxo-1-chlorophospholane (Cpt-Cl), hydroxybenzotriazole (HOBT),or mixtures thereof.

It has now been found that coupling of the bulky building units of thepresent invention may require the use of additional coupling reagentsincluding, but not limited to: coupling reagents such as PyBOP®(Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate), PyBrOP® (Bromo-tris-pyrrolidino-phosphoniumhexafluoro-phosphate), HBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), TBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate).

Novel coupling chemistries may be used, such as pre-formedurethane-protected N-carboxy anhydrides (UNCA's) and pre-formed acylfluorides. Said coupling may take place at room temperature and also atelevated temperatures, in solvents such as toluene, DCM(dichloromethane), DMF (dimethylformamide), DMA (dimethylacetamide), NMP(N-methyl pyrrolidinone) or mixtures of the above.

One object of the present invention is a method for the preparation ofbackbone cyclized peptide analogs of Formula (I): ##STR27## wherein thesubstituents are as defined above; comprising the steps of incorporatingat least one N.sup.α -ω-functionalized derivatives of amino acids ofFormula (VI): ##STR28##

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R' is an amino acid side chain such as H, CH₃, etc.,optionally bound with a specific protecting group; B is a protectinggroup selected from the group consisting of alkyloxy, substitutedalkyloxy, or aryloxy carbonyls; and G is a functional group selectedfrom the group consisting of amines, thiols, alcohols, carboxylic acidsand esters, aldehydes, alcohols and alkyl halides; and A is a specificprotecting group of G;

with a compound of the Formula (VII):

    H.sub.2 N--(AA).sub.f --CO--E                              Formula (VII)

wherein f is an integer from 1 to 10; (AA) designates an amino acidresidue wherein the amino acid residues may be the same or different,and E is a hydroxyl, a carboxyl protecting group or an amide to give acompound of the general Formula: ##STR29## (ii) selectively removingprotecting group B and reacting the unprotected compound with a compoundof Formula:

    B--NH--(AA).sub.e --COOH                                   Formula (IX)

wherein B and (AA) are as described above and e is an integer from 1 to10,

to give a compound of Formula: ##STR30##

wherein B, (AA), e, R¹, and f are as described above;

(iii) removing the protecting group B from the compound of the Formula(X) and reacting the unprotected compound with a compound of Formula:##STR31##

wherein X' is a spacer group selected from the group consisting ofalkylenes, substituted alkylenes, arylenes, cycloalkylenes andsubstituted alkylenes; G' is a functional group selected from amines,thiols, carboxyls, aldehydes or alcohols; A' is a specific-protectinggroup thereof; R¹ is an amino acid side chain such as H, CH₃, etc.,optionally bound with a specific protecting group; and B is a protectinggroup;

to yield a compound of Formula: ##STR32## (iv) removing the protectinggroup B and reacting the unprotected compound with a compound ofFormula:

    B--NH--(AA).sub.d --COOH                                   Formula (IXa)

to yield a compound of Formula: ##STR33## (v) selectively removingprotecting groups A and A' and reacting the terminal groups G and G' toform a compound of the Formula: ##STR34##

wherein d, e and f are independently an integer from 1 to 10; (AA) is anamino acid residue wherein the amino acid residues in each chain may bethe same or different; E is an hydroxyl group, a carboxyl protectinggroup or an amino group; R and R' are independently an amino acidside-chain such as H, CH₃, etc.; and the line designates a bridginggroup of the Formula: --X--M--Y--W--Z--

wherein M and W are independently selected from the group consisting ofdisulfide, amide, thioether, imine, ether, and alkene; X, Y and Z areindependently selected from the group consisting of alkylene,substituted alkylene, arylene, cycloalkylene, and substitutedcycloalkylene;

(vi) removing all remaining protecting groups to yield a compound ofFormula (I).

Bicyclic analogs are prepared in the same manner, that is, by repetitionof steps (v) and (vi). The determination of which residues are cyclizedwith which other residues is made through the choice of blocking groups.The various blocking groups may be removed selectively, thereby exposingthe selected reactive groups for cyclization.

Preferred are methods for the preparation of backbone cyclized peptideanalogs of Formula (I) wherein G is an amine, thiol or carboxyl group; Rand R' are each other than H, such as CH₃, (CH₃)₂ CH--, (CH₃)₂ CHCH₂ --,CH₃ CH₂ CH(CH₃)--, CH₃ S(CH₂)₂ --, HOCH₂ --, CH₃ CH(OH)--, HSCH₂ --, NH₂C(═O)CH₂ --, NH₂ C(═O)(CH₂)₂ --, HOC(═O)CH₂ --, HOC(═O)(CH₂)₂ --, NH₂(CH₂)₄ --, C(NH₂)₂ NH(CH₂)₃ --, HO--phenyl--CH₂ --, benzyl,methylindole, and methylimidazole, and wherein E is covalently bound toan insoluble polymeric support.

Another object of the present invention is a method for the preparationof backbone cyclized peptide analogs of Formula (II): ##STR35## whereinthe substituents are as defined above;

comprising the steps of: incorporating at least one ω-functionalizedamino acid derivative of the general Formula (VI): ##STR36##

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R is the side chain of an amino acid, such as H, CH₃,etc.; B is a protecting group selected from the group consisting ofalkyloxy, substituted alkyloxy, or aryloxy carbonyls; and G is afunctional group selected from the group consisting of amines, thiols,alcohols, carboxylic acids and esters or alkyl halides and A is aprotecting group thereof;

into a peptide sequence and subsequently selectively cyclizing thefunctional group with one of the side chains of the amino acids in saidpeptide sequence.

Preferred is the method for the preparation of backbone cyclized peptideanalogs of Formula (II) wherein G is a carboxyl group or a thiol group;R is CH₃, (CH₃)₂ CH--, (CH₃)₂ CHCH₂ --, CH₃ CH₂ CH(CH₃)--, CH₃ S(CH₂)₂--, HOCH₂ --, CH₃ CH (OH)--, HSCH₂ --, NH₂ C(═O)CH₂ --, NH₂ C(═O)(CH₂)₂--, HOC(═O)CH₂ --, HOC(═O)(CH₂)₂ --, NH₂ (CH₂)₄ --, C(NH₂)₂ NH(CH₂)₃ --,HO--phenyl--CH₂ --, benzyl, methylindole, and methylimidazole, andwherein E is covalently bound to an insoluble polymeric support.

Preparation of backbone to side chain cyclized peptide analogs isexemplified in Scheme I below. In this schematic example, the bridginggroup consists of alkylene spacers and an amide bond formed between anacidic amino acid side chain (e.g. aspartic or glutamic acid) and anω-functionalized amino acid having a terminal amine.

Scheme I

Preparation of peptides with Backbone to Side Chain cyclization.

One preferred procedure for preparing the desired backbone cyclicpeptides involves the stepwise synthesis of the linear peptides on asolid support and the backbone cyclization of the peptide either on thesolid support or after removal from the support. The C-terminal aminoacid is bound covalently to an insoluble polymeric support by acarboxylic acid ester or other linkages such as amides. An example ofsuch support is a polystyrene-co-divinyl benzene resin. The polymericsupports used are those compatible with such chemistries as Fmoc and Bocand include for example PAM resin, HMP resin and chloromethylated resin.The resin bound amino acid is deprotected for example with TFA to give(1) below and to it is coupled the second amino acid, protected on theN.sup.α for example by Fmoc, using a coupling reagent like BOP. Thesecond amino acid is deprotected to give (3) using for examplepiperidine 20% in DMF. The subsequent protected amino acids can then becoupled and deprotected at ambient temperature. After several cycles ofcoupling and deprotection that gives peptide (4), an amino acid havingfor example carboxy side chain is coupled to the desired peptide. Onesuch amino acid is Fmoc-aspartic acid t-butyl ester. After deprotectionof the N.sup.α Fmoc protecting group that gives peptide (5), the peptideis again elongated by methods well known in the art to give (6). Afterdeprotection a building unit for backbone cyclization (the preparationof which is described in Schemes III-VIII) is coupled to the peptideresin using for example the coupling reagent BOP to give (7). One suchbuilding unit is for example Fmoc-N.sup.α (ω-Boc-amino alkylene)aminoacid. After deprotection the peptide can then be elongated, to thedesired length using methods well known in the art to give (8). Thecoupling of the protected amino acid subsequent to the building unit isperformed by such coupling agents exemplified by PyBrOP® to ensure highyield.

After the linear, resin bound peptide, e.g. (8), has been prepared theω-alkylene-protecting groups for example Boc and t-Bu are removed bymild acid such as TFA to give (9). The resin bound peptide is thendivided into several parts. One part is subjected to on-resincyclization using for example TBTU as cyclization agent in DMF to ensurehigh yield of cyclization, to give the N-backbone to side chain cyclicpeptide resin (10). After cyclization on the resin the terminal aminoprotecting group is removed by agents such as piperidine and thebackbone to side chain cyclic peptide (11) is obtained after treatmentwith strong acid such as HF. Alternatively, prior to the removal of thebackbone cyclic peptide from the resin, the terminal amino group isblocked by acylation with agents such as acetic anhydride, benzoicanhydride or any other acid such as adamantyl carboxylic acid activatedby coupling agents such as BOP.

The other part of the peptide-resin (9) undergoes protecting of the sidechains used for cyclization, for example the ω-amino and carboxy groups.This is done by reacting the ω-amino group with for example Ac₂ O andDMAP in DMF and activating the free ω-carboxy group by for example DICand HOBT to give the active ester which is then reacted with for exampleDh₃ NH₂ to give the linear analog (13) of the cyclic peptide (10).Removal of the peptide from the resin and subsequent removal of the sidechains protecting groups by strong acid such as HF to gives (14) whichis the linear analog of the backbone to side chain cyclic peptide (11).

The linear analogs are used as reference compounds for the biologicalactivity of their corresponding cyclic compounds.

Reaction Scheme I Follows at this Point ##STR37##

The selection of N.sup.α and side chain protecting groups is, in part,dictated by the cyclization reaction which is done on the peptide-resinand by the procedure of removal of the peptide from the resin. TheN.sup.α protecting groups are chosen in such a manner that their removalwill not effect the removal of the protecting groups of the N.sup.α(ω-aminoalkylene) protecting groups. In addition, the removal of theN.sup.α (ω-aminoalkylene)protecting groups or any other protectinggroups on ω-functional groups prior to the cyclization, will not effectthe other side chain protection and/or the removal of the peptide fromthe resin. The selection of the side chain protecting groups other thanthose used for cyclization is chosen in such a manner that they can beremoved subsequently with the removal of the peptide from the resin.Protecting groups ordinarily employed include those which are well knownin the art, for example, urethane protecting substituents such as Fmoc,Boc, Alloc, Z and the like.

It is preferred to utilize Fmoc for protecting the α-amino group of theamino acid undergoing the coupling reaction at the carboxyl end of saidamino acid. The Fmoc protecting group is readily removed following suchcoupling reaction and prior to the subsequent step by the mild action ofbase such as piperidine in DMF. It is preferred to utilize Boc forprotecting ω-amino group of the N.sup.α (ω-aminoalkylene) group and t-Bufor protecting the carboxy group of the amino acids undergoing thereaction of backbone cyclization. The Boc and t-Bu protecting groups arereadily removed simultaneously prior to the cyclization.

Scheme II

Preparation of peptides with Backbone to Backbone cyclization.

Preparation of N-backbone to N-backbone cyclized peptide analogs isexemplified in scheme II. In this schematic example, the building groupconsists of alkylene spacers and two amide bonds.

A building unit for backbone cyclization (the preparation of whichdescribed in Schemes III-VIII) is coupled to a peptide resin, forexample peptide-resin (4), using for example the coupling reagent BOP togive (16). One such building unit is for example Fmoc-Nα(ω-Boc-aminoalkylene)amino acid. The side chain Boc protecting group is removed bymild acid such as TFA in DCM and an N-Boc protected ω-amino acid, or anyother Boc protected amino acid, is coupled to the side chain amino groupusing coupling agent such as BOP to give peptide-resin (17).

After deprotection of the N.sup.α Fmoc protecting group by mild basesuch as piperidine in DMF, the peptide can then be elongated, ifrequired, to the desired length using methods well known in the art togive (18). Alternatively, the deprotection of the N.sup.α Fmoc andsubsequent elongation of the peptide can be done before deprotection ofthe side chain Boc protecting group. The elongation of the N-alkyleneside chain allow control of the ring size. The coupling of the protectedamino acid subsequent to the building unit is performed by such couplingagents exemplified by PyBrOP® to ensure high yield.

After deprotection of the terminal N.sup.α Fmoc group, a second buildingunit, for example Fmoc-N.sup.α (ω-t-Bu-carboxy-alkylene)amino acid iscoupled to the peptide-resin to give (19). After deprotection of theN.sup.α Fmoc protecting group, the peptide can then be elongated, ifrequired, to the desired length using methods well known in the art togive (20). The coupling of the protected amino acid subsequent to thebuilding unit is performed by such coupling agents exemplified byPyBrOP® to ensure high yield. After the linear, resin bound peptide,e.g. (20), has been prepared the ω-alkylene-protecting groups forexample Boc and t-Bu are removed by mild acid such as TFA to give (21).The resin peptide is then divided into several parts. One part issubjected to on-resin cyclization using for example TBTU as cyclizationagent in DMF to ensure high yield of cyclization, to give the N-backboneto N-backbone cyclic peptide resin (22). After cyclization on the resinthe terminal amino protecting group is removed by agents such aspiperidine and the backbone to backbone cyclic peptide (23) is obtainedafter treatment with strong acid such as HF. Alternatively, prior to theremoval of the backbone cyclic peptide from the resin, the terminalamino group of (22) is blocked, after deprotection, by acylation withagents such as acetic anhydride, benzoic anhydride or any other acidsuch as adamantyl carboxylic acid activated by coupling agents such asBOP to give the N-terminal blocked backbone to backbone cyclic peptide(24).

The other part of the peptide-resin (21) undergoes protecting of theside chains used for cyclization, for example the ω-amino and carboxygroups. This is done by reacting the ω-amino group with for example Ac₂O and DMAP in DMF and activating the free ω-carboxy group by for exampleDIC and HOBT to give the active ester which is then reacted with forexample MeNH₂. Removal of the peptide from the resin and subsequentremoval of the side chains protecting groups by strong acid such as HFto gives (26) which is the linear analog of the backbone to backbonecyclic peptide (23). The linear analogs are used as reference compoundsfor the biological activity of their corresponding cyclic compounds.

Reaction Scheme II Follows at this Point ##STR38## Novel Synthesis ofBuilding Units

The novel synthesis providing N(ω-(functionalized) alkylene) amino acidsused to generate backbone cyclic peptides is depicted in schemesIII-VIII. In this approach we have implemented the following changes inorder to devise a practical, general synthesis:

1. The nucleophile is a secondary nitrogen, which is a betternucleophile than the primary nitrogen previously used. This alsoprevents the possibility of double alkylation.

2. The leaving group was changed to trifluoromethanesulfonyl (triflate),which has a much lower tendency to eliminate than a halogen, thus makingit possible to implement the synthesis with amino acids other thanglycine. Furthermore, the triflate leaving group prevents racemizationduring the alkylation reaction.

3. The carboxylate is esterified prior to the substitution reaction, tofacilitate the substitution by removing the negative charge next to theelectrophilic carbon.

Scheme III

Preparation of N.sup.α, N.sup.ω protected ω-amino alkylene amino acidsbuilding units.

One preferred procedure for the preparation of protected N.sup.α(ω-amino alkylene) amino acids involves the N.sup.α alkylation ofsuitably protected diamino alkanes. One preferred N.sup.α,N.sup.ω di-protected diamino alkane is for example N.sup.α -Benzyl, N.sup.ω -Bocdiamino alkane (27). This starting material contains one protectinggroup such as Boc which is necessary for the final product, and atemporary protecting group such as Bzl to minimize unwanted sidereactions during the preparation of the titled compound. One preferredprocedure for the preparation of the starting material (27) involvesreductive alkylation of N-Boc diamino alkane with aldehydes such asbenzaldehyde. The temporary protection of the N.sup.α amino group, whichis alkylated in the reaction by such protecting groups as Bzl, minimizesthe dialkylation side reaction and allows removal by such conditionsthat do not remove the N.sub.ω -protecting group.

The N.sup.α,N.sup.ω di-protected diamino alkane is reacted with forexample chiral α-hydroxy α-substituted acid esters where the hydroxylmoiety is converted to a leaving group for example Triflate.

The use of Triflate as the leaving group was found to be superior toother leaving groups such as halogens, Tosyl, Mesyl, etc., because itprevents the β-elimination reaction encountered with the other leavinggroups. The use of Triflate as the leaving group also ensures highoptical purity of the product (28). The temporary N.sup.α protectinggroup, such as Bzl, and the carboxyl protecting group, such as methylester, are removed by mild conditions, such as catalytic hydrogenationand hydrolysis, that do not remove the N.sup.ω protecting group such asBoc to give the N.sup.ω protected amino acid (29). Introduction of theN.sup.α protecting group suitable for peptide synthesis is accomplishedby methods well known in the art, to give the protected N.sup.α (N.sup.ωprotected amino alkylene) amino acid (30).

The choice of the N.sup.α and the N.sup.ω protecting groups is dictatedby the use of the building units in peptide synthesis. The protectinggroups have to be orthogonal to each other and orthogonal to the otherside chains protecting groups in the peptide. Combinations of N.sup.αand N.sup.ω protecting groups are for example: N.sup.α -Fmoc, N.sup.ω-Boc; N.sup.α -Fmoc, N.sup.ω -Alloc; N.sup.α -Boc, N.sup.ω -Alloc. Thesecombinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution.

Scheme IV

Preparation of N.sup.α, N.sup.ω protected ω-amino alkylene glycinebuilding units.

One preferred procedure for the preparation of protected N.sup.α(ω-amino alkylene) glycines involves the reaction of the N.sup.α,N.sup.ω di-protected di amino alkane (27) with commercially availableα-activated carboxylic acid esters, for example benzylbromo acetate.Since the titled compound is achiral, the use of leaving groups such asTrf, Tos or Mes is not necessary. The use of the same temporaryprotecting groups for the N.sup.α and the carboxy groups, for examplethe Bzl protecting group, ensures the prevention of the undesireddialkylation side reaction and allows concomitant removal of thetemporary protecting groups thus giving high yield of the N.sup.ωprotected amino acid (32). Introduction of the N.sup.α protecting groupsuitable for peptide synthesis is accomplished by methods well known inthe art, to give the protected N.sup.α (N.sup.ω protected aminoalkylene) glycines (33).

The choice of the N.sup.α and the N.sup.ω protecting groups is dictatedby the use of the building units in peptide synthesis. The protectinggroups have to be orthogonal to each other and orthogonal to the otherside chains protecting groups in the peptide. Combinations of N.sup.αand N.sup.ω protecting groups are for example: N.sup.α Fmoc, N.sup.ω Boc; N.sup.α Fmoc, N.sup.ω Alloc; N.sup.α Boc, N.sup.ω Alloc. Thesecombinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution. ##STR39##

Scheme V

Preparation of N.sup.α, ω-carboxy protected ω-carboxy alkylene aminoacids.

One preferred procedure for the preparation of protected N.sup.α(ω-carboxy alkylene) amino acids involves the N.sup.α -alkylation ofsuitably N.sup.α, ω-carboxy deprotected amino acids. One preferreddeprotected amino acid is N.sup.α -Benzyl ω-amino acids t-butyl esters(34). This starting material contains one protecting group such as t-Buester which is necessary for the final product, and a temporaryprotecting group such as N.sup.α Bzl to minimize side reactions duringthe preparation of the titled compound. One preferred procedure for thepreparation of the starting material (34) involves reductive alkylationof ω-amino acids t-butyl esters with aldehydes such as benzaldehyde. Thetemporary protection of the amino group which is used as nucleophile inthe proceeding alkylation reaction by such protecting groups as Bzlminimizes the dialkylation side reaction.

The N.sup.α, ω-carboxy deprotected amino acids (34) are reacted with,for example, chiral α-hydroxy α-substituted acid esters where thehydroxyl moiety is converted to a leaving group, for example, Triflate.The use of Triflate as the leaving group was found to be superior toother leaving groups such as halogens, Tosyl, Mesyl; etc., because itprevents the β-elimination reaction encountered with the other leavinggroups. The use of Triflate as the leaving group also ensures highoptical purity of the product, for example (36). The temporary N.sup.αprotecting group, such as Bzl, and the α-carboxyl protecting group, suchas benzyl ester, are concomitantly removed by mild condition, such ascatalytic hydrogenation, that to not remove the ω-carboxy protectinggroup such as t-Bu to give the N.sup.α (protected ω-carboxy alkylene)amino acid (36). Introduction of the N.sup.α protecting group suitablefor peptide synthesis is accomplished by methods well known in the art,to give the protected N.sup.α (ω protected carboxy alkylene) amino acid(37).

The choice of the N.sup.α and the ω-carboxy protecting groups isdictated by the use of the building units in peptide synthesis. Theprotecting groups have to be orthogonal to each other and orthogonal tothe other side chains protecting groups in the peptide. A combination ofN.sup.α and ω-carboxy protecting groups are for example: N.sup.α -Fmoc,ω-carboxy t-Bu; N.sup.α -Fmoc, ω-carboxy Alloc; N.sup.α -Boc, ω-carboxyAlloc. These combinations are suitable for peptide synthesis andbackbone cyclization, either on solid support or in solution.

Scheme VI

Preparation of N.sup.α, ω-carboxy protected ω-carboxy alkylene glycinebuilding units.

One preferred procedure for the preparation of protected N.sup.α(ω-carboxy alkylene)glycines involves the N.sup.α -alkylation ofsuitably N.sup.α, ω-carboxy deprotected amino acids (34) withcommercially available α-activated carboxylic acid esters for example,benzyl bromo acetate. Since the titled compound is achiral, the use ofleaving groups such as Trf, Tos or Mes is not necessary.

The use of the same temporary protecting groups for the N.sup.α and theα-carboxy groups, for example the Bzl protecting group, ensures theprevention of the undesired dialkylation side reaction and allowsconcomitant removal of the temporary protecting groups thus giving highyield of the N.sup.α (protected ω-carboxy alkylene) glycines (39).Introduction of the N.sup.α protecting group suitable for peptidesynthesis is accomplished by methods well known in the art, to give theprotected N.sup.α (ω protected carboxy alkylene) glycines (40).

The choice of the N.sup.α and the ω-carboxy protecting groups isdictated by the use of the building units in peptide synthesis. Theprotecting groups have to be orthogonal to each other and orthogonal tothe other side chains protecting groups in the peptide. A combination ofN.sup.α and ω-carboxy protecting groups are, for example: N.sup.α Fmoc,ω-carboxy t-Bu; N.sup.α Fmoc, ω-carboxy Alloc; N.sup.α Boc, ω-carboxyAlloc. These combinations are suitable for peptide synthesis andbackbone cyclization, either on solid support or in solution. ##STR40##

Scheme VII

Preparation of N.sup.α S.sup.ω protected ω-thio alkylene amino acidbuilding units.

One preferred procedure for the preparation of N.sup.α, S.sup.ω-deprotected N.sup.α (ω-thio alkylene) amino acids involves the N.sup.α-alkylation of suitably S.sup.ω protected ω-thio amino alkanes. SuitableS.sup.ω protecting groups are, for example, Bzl, t-Bu, Trt. Onepreferred S.sup.ω -protected ω-thio amino alkanes is for exampleω-(S-Benzyl) amino alkanes (41). One preferred procedure for thepreparation of the starting material (41) involves the use of salts ofS-protected thiols as nucleophiles for a nucleophilic substitutionreaction on suitably N.sup.α -protected ω-activated amino alkanes.Removal of the amino protection gives the starting material (41).

The S-protected ω-thio amino alkanes (41) are reacted with for examplechiral α-hydroxy α-substituted acid esters where the hydroxyl moiety isconverted to a leaving group for example Triflate. The use of Triflateas the leaving group was found to be superior to other leaving groupssuch as halogens, Tosyl, Mesyl etc. because it prevents theβ-elimination reaction encountered with the other leaving groups. Theuse of Triflate as the leaving group also ensures high optical purity ofthe product for example (42). The temporary α-carboxyl protecting group,such as methyl ester, is removed by mild condition, such as hydrolysiswith base, that to not remove the ω-thio protecting group such as S-Bzlto give the N.sup.α (S-protected ω-thio alkylene) amino acid (43).Introduction of the N.sup.α protecting group suitable for peptidesynthesis is accomplished by methods well known in the art, to give theprotected N,S protected N.sup.α (ω-thio alkylene) amino acid (44).

The choice of the N.sup.α and the ω-thio protecting groups is dictatedby the use of the building units in peptide synthesis. The protectinggroups have to be orthogonal to each other and orthogonal to the otherside chains protecting groups in the peptide. A combination of N.sup.αand ω-thio protecting groups are for example: N.sup.α Fmoc, S.sup.ωt-Bu; N.sup.α Fmoc, S.sup.ω Bzl; N.sup.α Fmoc, S.sup.ω Trt; N.sup.α Boc,S.sup.ω Bzl. These combinations are suitable for peptide synthesis andbackbone cyclization, either on solid support or in solution.

Scheme VIII

Preparation of N.sup.α, S.sup.ω protected ω-thio alkylene glycinebuilding units.

One preferred procedure for the preparation of N.sup.α, S.sup.ω-deprotected N.sup.α (ω-thio alkylene) amino acids involves the N.sup.α-alkylation of suitably S.sup.ω protected ω-thio amino alkanes (41) withcommercially available α-activated carboxylic acid esters for exampleethyl bromo acetate. Since the titled compound is achiral, the use ofleaving groups such as Trf, Tos or Mes is not necessary.

Suitable protecting groups for the ω-thio groups are for example Bzl,t-Bu, Trt. One preferred S-protected ω-thio amino alkanes is for exampleω-(S-Benzyl) amino alkanes (41). The N-alkylation reaction gives theester (45). The temporary α-carboxyl protecting group, such as ethylester, is removed by mild conditions, such as hydrolysis with base, thatto not remove the ω-thio protecting group such as S-Bzl to give theN.sup.α (S-protected ω-thio alkylene) glycines (46). Introduction of theN.sup.α protecting group suitable for peptide synthesis is accomplishedby methods well known in the art, to give the protected N.sup.α, S.sup.ω-deprotected N.sup.α (ω-thio alkylene) glycines (47).

The choice of the N.sup.α and the ω-thio protecting groups is dictatedby the use of the building units in peptide synthesis. The protectinggroups have to be orthogonal to each other and orthogonal to the otherside chains protecting groups in the peptide. A combination of N.sup.αand ω-thio protecting groups are for example: N.sup.α Fmoc, S.sup.ωt-Bu; N.sup.α Fmoc, S.sup.ω Bzl; N.sup.α Fmoc, S.sup.ω Trt; N.sup.α Boc,S.sup.ω Bzl. These combinations are suitable for peptide synthesis andbackbone cyclization, either on solid support or in solution. ##STR41##

SPECIFIC EXAMPLES OF PEPTIDES

Preparation of the novel backbone cyclized peptide analogs using theschematics outlined above will be illustrated by the followingnon-limiting specific examples:

Example 1

Ada-(D)Arg-Arg-cyclo(N.sup.α(1-(6-aminohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg--OH

Stage 1

Soc-Arg(Tos)-O-resin→Fmoc-Phe-Arg(Tos)-O-resin

Boc-L-Arg(Tos)-O-resin (0.256 g, 0.1 mmole, 0.39 meq of nitrogen/g) wasplaced in a shaker flask and swelled for two hours by the addition ofDCM. The resin was then carried out through the procedure in Table 1which includes two deprotections of the Boc protecting group with 55%TFA in DCM for a total of 22 minutes, washing, neutralization with 10%DIEA in NMP and washing (Table 1 steps 1-8). After positive ninhydrintest, as described in Kaiser et al., Anal Biochem., 34:595, 1970 and isincorporated herein by reference in its entirety, coupling (Table 1steps 9-10) was achieved in NMP by the addition of Fmoc-L-Phe (0.232 g,0.6 mmole) and after 5 minutes of shaking, solid BOP reagent (0.265 g,0.6 mmole) was added to the flask.

                  TABLE 1    ______________________________________    PROCEDURE FOR 0.1 mMOLE SCALE    STEP SOLVENT/  VOLUME   TIME  REPEAT    NO.  REAGENT   (ML)     (MIN) (XS)   COMMENT    ______________________________________    1    DCM       5        120   1      Swells resin    2    DCM       5        2     3    3    TFA/      5        2     1      Deprotection         DCM 55%    4    TFA/      5        20    1      Deprotection         DCM 55%    5    DCM       5        2     3    6    NMP       5        2     4      check for                                         positive nin.    7    DIEA/NMP  5        5     2      Neutralization    8    NMP       5        2     5    9    Fmoc-AA            5     5      Coupling add         in NMP                          BOP 6 eq. add                                         DIEA 120 600 1                                         12 eq. Check                                         pH, adjust to                                         pH 8 with DIEA    10   NMP       5        2     5      check for                                         negative nin.    11   Pip/      5        10    1      Deprotection         NMP 20%    12   Pip/      5        10    1         NMP 20%    13   NMP       5        2     6      check for                                         positive nin.    ______________________________________

After shaking for 10 minutes, the mixture was adjusted to pH 8 (measuredwith wetted pH stick) by the addition of DIEA (0.209 mL, 1.2 mmole) andthe flask shaken for 10 hours at ambient temperature. The resin was thenwashed and subjected to ninhydrin test. After negative ninhydrin testthe resin was used for the next coupling.

Stage 2

Fmoc-Phe-Arg(Tos)-O-resin→Fmoc-N.sup.α (6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin

The Fmoc-Phe-Arg(Tos)-O-resin (Stage 1) was subjected to twodeprotections of the Fmoc protecting group by 20% Pip in NMP (Table 1steps 11-13). After washing and ninhydrin test, coupling of Fmoc-D-Phewas achieved as described in Stage 1 (Table 1 steps 9-10) usingFmoc-D-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) andDIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc groupdeprotected as described above (Table 1 steps 11-13). After washing andninhydrin test, coupling of Fmoc-D-Asp(t-Bu) was achieved as describedin Stage 1 (Table 1 steps 9-10) using Fmoc-D-Asp(t-Bu) (0.247 g, 0.6mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole).The resin was washed and the Fmoc group deprotected as described above(Table 1 steps 11-13). After washing and ninhydrin test, coupling ofFmoc-L-Phe was achieved as described in Stage 1 (Table 1 steps 9-10)using Fmoc-L-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole)and DIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc groupdeprotected as described above (Table 1 steps 11-13). After washing andninhydrin test, coupling of Fmoc-L-Hyp(OBzl) was achieved as describedin Stage 1 (Table 1 steps 9-10) using Fmoc-L-Hyp(OBzl) (0.266 g, 0.6mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole).The resin was washed and the Fmoc group deprotected as described above(Table 1 steps 11-13). The resin was washed and subjected to picric acidtest. Coupling of Fmoc-N.sup.α (6-Boc amino hexylene) glycine wasachieved as described in Stage 1 (Table 1 steps 9-10) using Fmoc-N.sup.α(6-Boc amino hexylene)glycine (0.3 g, 0.6 mmole), BOP reagent (0.265 g,0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was then washed andsubjected to the picric acid test. After negative test the resin wasused for the next coupling.

Stage 3

Fmoc-N.sup.α (6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin→Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup.α (6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin

The Fmoc-N.sup.α (6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin (Stage2) was subjected to three deprotection of the Fmoc protecting group by20% Pip in NMP (Table 2 steps 1-2). After washing, the picric acid testwas performed. If the test did not show 98±2%, deprotection of thepeptide resin was subjected again to 3 deprotection steps (Table 2 steps1-2), washing and picric acid test. Coupling of Fmoc-L-Arg(Tos) wasachieved in NMP by the addition of (0.33 g, 0.6 mmole) and after 5minutes of shaking, solid PyBOP reagent (0.28 g, 0.6 mmole) was added tothe flask. After shaking for 10 minutes, the mixture was adjusted to pH8 (measured with wetted pH stick) by the addition of DIEA (0.209 mL, 1.2mmole) and the flask shaken for 2.5 hours at ambient temperature. Theresin was then washed and subjected to a second coupling by the sameprocedure for 20 hours. After washing the resin was subjected to picricacid test (Table 2 steps 3-6). If the test did not show 98±2% couplingthe peptide resin was subjected again to a third coupling for 2 hours at50° C. (Table 2 step 7). The resin was washed subjected to threedeprotection of the Fmoc protecting group by 20% Pip in NMP (Table 2steps 1-2). After washing picric acid test was performed.

                  TABLE 2    ______________________________________    PROCEDURE FOR 0.1 mMOLE SCALE    STEP SOLVENT/  VOLUME   TIME  REPEAT    NO.  REAGENT   (ML)     (MIN) (XS)   COMMENT    ______________________________________    1    Piperidine/                   5        10    3      Deprotection         NMP 20%    2    NMP       5        2     6      Picric acid test.    3    Fmoc-AA   5        5            Coupling         in NMP         add PyBroP                      6 eq.         add DIEA           150   1      12 eq. Check pH,                                         adjust to pH 8                                         with DIEA.    4    NMP       5        2     3      check for nega-                                         tive nin.    5    Fmoc-AA   5        5            Coupling         in NMP         add PyBroP                      6 eq.         add DIEA           20 hr.                                  1      12 eq.                                         Check pH, adjust                                         to pH 8 with                                         DIEA.    6    NMP       5        2     4      Picric acid test.                                         If less than                                         98 ± 2%                                         coupling repeat                                         Steps 4-5    7    Fmoc-AA   5        5            Coupling at         in NMP                          50° C.         add PyB0P                       6 eq.         add DIEA           120   1      12 eq.                                         Check pH, adjust                                         to pH 8 with                                         DIEA.    8    NMP       5        2     4    ______________________________________

If the test did not show 98±2% deprotection, the peptide resin wassubjected again to 3 deprotection steps (Table 2 steps 1-2), washing andthe picric acid test. Coupling of Fmoc-D-Arg(Tos) was achieved in NMP asdescribed in Stage 1 (Table 1 steps 9-10) using Fmoc-D-Arg(Tos) (0.33 g,0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2mmole). The resin was washed 6 times with NMP (Table 1 step 15) and usedin the next stages.

Stage 4

Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup.α (6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin→Ada-D-Arg-Arg-cyclo(N.sup.α(1-(6-amidohexylene) Gly-Hyo-Phe-D-Asp)-D-Phe-Phe-Arg--OH

The Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup.α (6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin (Stage3) was subjected to deprotection of the Boc and t-Bu protecting groupsand on resin cyclization according to Table 3. The peptide resin waswashed with DCM and deprotected as described in Stage 1 by 55% TFA inDCM. After washing and neutralization by 10% DIEA in NMP and washing 6times with DCM the peptide resin was dried in vacuo for 24 hours. Thedry peptide resin weight, 0.4 g, it was divided into two parts. 0.2 g ofthe peptide resin was swollen 2 hours in 5 mL NMP and cyclized asfollows: Solid TBTU reagent (0.19 g, 6 mmole) was added to the flask.After shaking for 10 minutes, the mixture was adjusted to pH 8 by theaddition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5hours at ambient temperature. The resin was then washed and subjected toa second coupling by the same procedure for 20 hours. After washing theresin was subjected to picric acid test (Table 3 steps 8-11). If thetest did not show 98±2% cyclization the peptide resin was subjectedagain to a third cyclization for 2 hours at 50° C. (Table 2 step 12).The resin was washed, subjected to three deprotection of the Fmocprotecting group by 20% Pip in NMP (Table 2 steps 1-2). After washingand ninhydrin test, the N-terminal amino group was blocked by Ada.Adamantane acetic acid (0.108 g, 6 mmole), BOP reagent (0.265 g, 0.6mmole) and DIEA (0.209 mL, 1.2 mmole) were added and the flask shakenfor 2 hours. After washing 6 times with NMP (Table 2 step 13), ninhydrintest was performed. If the test was positive or slightly positive theprotecting with adamantane acetic acid was repeated. If the ninhydrintest was negative, the peptide resin was washed 6 times with NMP and 6times with DCM. The resin was dried under vacuum for 24 hours. The driedresin was subjected to HF as follows: to the dry peptide resin (0.2 g)in the HF reaction flask, anisole (2 mL) was added and the peptidetreated with 20 mL liquid HF at -20° C. for 2 hours. After theevaporation of the HF under vacuum, the anisole was washed with ether(20 mL, 5 times) and the solid residue dried in vacuum. The peptide wasextracted from the resin with TFA (10 mL, 3 times) and the TFAevaporated under vacuum. The residue was dissolved in 20 mL 30% AcOH andlyophilized. This process was repeated 3 times. The crude peptide waspurified by semiprep HPLC. The final product was obtained as whitepowder by lyophilization from dioxane, which gave 42 mg (56%) of thetitle compound.

HPLC RT 32.15 minutes, 95%

TOF MS: 1351.4 (M⁺)

AAA in agreement with the title compound

Table 3 follows at this

                  TABLE 3    ______________________________________    PROCEDURE FOR 0.05 mMOLE SCALE    STEP SOLVENT/  VOLUME   TIME  REPEAT    NO.  REAGENT   (ML)     (MIN) (XS)   COMMENT    ______________________________________    1    DCM       5        2     3    2    TFA/DCM   5        2     1      Deprotection         55%    3    TFA/DCM   5        20    1      Deprotection         55%    4    DCM       5        2     3    5    NMP       5        2     4    6    DIEA/NMP  5        5     2      Neutralization         10%    7    NMP       5        2     5    8    TBTU/NMP/ 5        150   3      Cyclization         DIEA    9    NMP       5        2     4      Picric acid test.                                         If less than                                         98 ± 2%                                         coupling perform                                         Steps 10-12. If                                         above 98 ± 2%,                                         go to step 13.    10   TBTU/NMP/ 5        20 hr 3      Cyclization.         DIEA                            Check pH, adjust                                         to pH 8                                         with DIEA.    11   NMP       5        2     4      Picric acid test.                                         If less than                                         98 ± 2%                                         coupling perform                                         Steps 12.                                         If above 98 ± 2%,                                         go to step 13    12   TBTU/NMP/ 5        120   3      Cyclization, 50 C.         DIEA                            Check pH, adjust                                         to pH 8 with                                         DIEA.    13   NMP       5        2     6    14   Pip/NMP   5        10    1      Deprotection         20%    15   Pip/NMP   5        10    1         20%    16   NMP       5        2     6      Check for posi-                                         tive nin.    17   AdacOH/   5        2     1         BOP/NMP    18   NMP       5        2     6      Check for nega-                                         tive nin.    19   DCM       5        2     4    ______________________________________

Example 2

NON-CYCLIZED PEPTIDE (Control for biological assays)

Ada-D-Arg-Arg-N.sup.α(6-acetamidohexylene)Gly-Hyp-Phe-D-Asp(NH-Me)-D-Phe-Phe-Arg--OH

The Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup.α (6-aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp-D-Phe-Phe-Arg(Tos)-O-resin (0.2 g)which was prepared in Example 1 Stage 4 was subjected to acetylation ofthe 6-amino side chain of N.sup.α (6-acetamidohexylene)Gly and to methylamidation of the carboxylic group of D-Asp as described in Table 4. Thepeptide resin was swollen in 5 mL NMP for 2 hours and AcO (0.113 mL, 12mmole) and PP (17 mg) were added. After 30 minutes, the resin was washedwith NMP 6 times and subjected to ninhydrin test. If the test waspositive or slightly positive the acetylation reaction was repeated. Ifthe ninhydrin test was negative, the carboxy group of D-Asp wasactivated by the addition of HOBT (0.040 g, 0.3 mmole) and DIC (0.047mL, 0.3 mmole) to the peptide resin in NMP. The mixture was shaken forhalf an hour and a solution of 30% methylamine in EtOH (0.2 mL) wasadded. After one hour, the resin was washed 6 times with NMP and theterminal Fmoc group removed by 20% Pip in NMP (Table 4 steps 7-9). Afterwashing with NMP the N-terminal amino group was blocked by Ada asdescribed in Example 1 Stage 4 and the resin was washed with NMP and DCM(Table 4 steps 10-12) and the resin dried in vacuo. The peptide wasdeprotected and cleaved from the resin by HF. To the dry peptide resin(0.2 g) in the HF reaction flask, anisole (2 mL) was added and thepeptide treated with 20 mL liquid HF at -20° C. for 2 hours. After theevaporation of the HF under vacuum, the anisole was washed with ether(20 mL 5 times) and the solid residue dried in vacuo. The peptide wasextracted from the resin with TFA (10 mL, 3 times) and the TFAevaporated under vacuum. The residue was dissolved in 20 mL 30% AcOH andlyophilized. This process was repeated 3 times. The crude peptide waspurified by semiprep HPLC. The final product was obtained as whitepowder by lyophilization from dioxane, which gave 48 mg (64%) of thetitle compound.

HPLC RT 27.70 minutes, 93%

TOF MS: 1424.6 (M⁺)

AAA in agreement with the title compound

                  TABLE 4    ______________________________________    PROCEDURE FOR 0.05 mMOLE SCALE    STEP SOLVENT/  VOLUME   TIME  REPEAT    NO.  REAGENT   (ML)     (MIN) (XS)   COMMENT    ______________________________________    1    NMP       5        120   1      Swells resin    2    Ac.sub.2 O/PP/                   5        30    1      Protecting of side         NMP                             chain    3    NMP       5        2     6      Check for nega-                                         tive nin.    4    DIC/HOBT/ 5        30    1      Activation of         NMP                             COOH side chain    5    MeNH.sub.2 /                   5        60    1      Protecting of side         EtOH/                           chain    6    NMP       5        2     6    7    Pip/NMP   5        10    1      Deprotection         20%    8    Pip/NMP   5        10    1         20%    9    NMP       5        2     6      Check for posi-                                         tive nin.    10   AdacOH/   5        2     1         BOP/NMP    11   NMP       5        2     6      Check for nega-                                         tive nin.    12   DCM       5        2     4    ______________________________________

Example 3

H-D-Arg-Arg-cyclo(N.sup.α (1-(4-propanoyl))Gly-Hyp-Phe-N.sup.α(3-amido-propylene)Gly)-Ser-D-Phe-Phe-Arg--OH

Stage 1

Fmoc-Phe-Arg(Tos)-O-resin→Fmoc-N.sup.α(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup.α (3-Boc aminopropylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin

Fmoc-Phe-Arg(Tos)-O-resin prepared from Boc-Arg(Tos)-O-Resin (0.3 g, 0.1mmole) (Example 1, Stage 1) was subjected to two deprotection of theFmoc protecting group by 20% piperidine in NMP (Table 1, steps 11-13).After washing and ninhydrin test, coupling of Fmoc-D-Phe was achieved asdescribed in Stage 1 (Example 1) (Table 1 steps 9-10) using Fmoc-D-Phe(0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209mL, 1.2 mmole). The resin was washed and the Fmoc group deprotected asdescribed above (Table 1, steps 11-13). After washing and ninhydrintest, coupling of Fmoc-Ser(BzL) was achieved as described in Stage 1(Example 1) (Table 1 steps 9-10) using Fmoc-Ser(Bzl) (0.25 g, 0.6mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole).The resin was washed and the Fmoc group deprotected as described above(Table 1, steps 11-13). After washing and picric acid test, coupling ofFmoc-N.sup.α (3-Boc amino propylene)glycine was achieved as described inTable 1, steps 9-10 using Fmoc-N.sup.α (3-Boc amino propylene)Gly (0.272g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2mmole). The resin was washed and subjected to three deprotection of theFmoc protecting group by 20% Pip in NMP (Table 2, steps 1-2). Afterwashing picric acid test was performed. If the test did not show 98±2%deprotection the peptide resin was subjected again to 3 deprotectionsteps (Table 2, steps 1-2), washing and picric acid test. Coupling ofFmoc-L-Hyp(OBzl) was achieved in NMP by the addition of Fmoc-L-Hyp(OBzl)(0.33 g, 0.6 mmole) and after 5 minutes of shaking, solid PyBrOP reagent(0.28 g, 0.6 mmole) was added to the flask. After shaking for 10minutes, the mixture was adjusted to pH 8 by the addition of DIEA (0.209mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambienttemperature. The resin was then washed and subjected to a secondcoupling by the same procedure for 20 hours. After washing the resin wassubjected to picric acid test (Table 2, steps 3-6). If the test did notshow 98±2% coupling the peptide resin was subjected again to a thirdcoupling for 2 hours at 50° C. (Table 2, step 7). The resin was washedsubjected to three deprotection of the Fmoc protecting group by 20% Pipin NMP (Table 2, steps 1-2). After washing picric acid test wasperformed. If the picric acid test did not show 98±2% deprotection, theresin was subjected again to deprotections steps (Table 2, steps 1-2).Coupling of Fmoc-Phe was achieved in NMP by the addition of Fmoc-Phe(0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209mL, 1.2 mmole). The resin was washed and after picric acid test the Fmocgroup deprotected as described above (Table 2, steps 1-2). After washingpicric acid test was performed. If the test did not show 98±2%deprotection the peptide resin was subjected again to 3 deprotectionsteps (Table 2, steps 1-2), washing and picric acid test. Coupling ofN.sup.α (3-t-Bu carboxy propylene)Gly was achieved as described in Table1 steps 9-10 using N.sup.α (3-t-Bu carboxy propylene)Gly (0.264 g, 0.6mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole).The resin was then washed and subjected to the picric acid test. Afternegative test the resin was used for the next coupling.

Stage 2

Fmoc-N.sup.α (4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup.α (3-Boc aminopropylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin→Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup.α(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup.α (3-Boc aminopropylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin

Fmoc-N.sup.α (4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup.α (3-Boc aminopropylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 1) wassubjected to three deprotection of the Fmoc protecting group by 20%Piperidine in NMP (Table 2, steps 1-2). After washing picric acid testwas performed. If the test did not show 98±2% deprotection the peptideresin was subjected again to 3 deprotection steps, washing, and picricacid test. Coupling of Fmoc-L-Arg(Tos) was achieved in NMP by theaddition of (0.33 g, 0.6 mmole) and after 5 minutes of shaking, solidPyBroP reagent (0.28 g, 0.6 mmole) was added to the flask. After shakingfor 10 minutes, the mixture was adjusted to pH 8 by the addition of DIEA(0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambienttemperature. The resin was then washed and subjected to a secondcoupling by the same procedure for 20 hours. After washing the resin wassubjected to picric acid test (Table 2, steps 3-6). If the test did notshow 98±2% coupling the peptide resin was subjected again to a thirdcoupling for 2 hours at 50° C. (Table 2, step 7). The resin was washedsubjected to three deprotection of the Fmoc protecting group by 20% Pipin NMP (Table 2, steps 1-2). After washing picric acid test wasperformed. If the test did not show 98±2% deprotection the peptide resinwas subjected again to 3 deprotection steps (Table 2, steps 1-2),washing and picric acid test. Coupling of Fmoc-D-Arg(Tos) was achievedin NMP as described in Stage 1 (Table 1, steps 9-10) usingFmoc-D-Arg(Tos) (0.33 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole)and DIEA (0.209 mL, 1.2 mmole). The resin was washed 6 times with NMP(Table 1, step 15) and used in the next stages.

Stage 3

Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup.α(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup.α (3-Bocamino-propylene)Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin→H-D-Arg-Arg-cyclo(N.sup.α(4-propanoyl))Gly-Hyp-Phe-N.sup.α(3-amido-propyl)Gly)-Ser-D-Phe-Phe-Arg--OH

Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup.α(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup.α (3-Bocamino-propylene)Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 2) wassubjected to deprotection of the Boc and t-Bu protecting groups and onresin cyclization according to Table 5. The peptide resin was washedwith DCM and deprotected as described in Stage 1 by 55% TFA in DCM.After washing and neutralization by 10% DIEA in NMP and washing 6 timeswith and NMP (Table 5, steps 1-5) the peptide was cyclized as follow:solid TBTU reagent (0.19 g, 6 mmole) was added to the flask. Aftershaking for 10 minutes, the mixture was adjusted to pH 8 by the additionof DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours atambient temperature. The resin was then washed and subjected to a secondcoupling by the same procedure for 20 hours. After washing the resin wassubjected to picric acid test (Table 3, steps 8-11). If the test did notshow 98±2% cyclization the peptide resin was subjected again to a thirdcyclization for 2 hours at 50° C. (Table 2, step 12). The resin waswashed, subjected to three deprotection of the Fmoc protecting group by20% Pip in NMP (Table 5, steps 14-15). After washing 6 times with NMPand 4 times with DCM, the resin was dried in vacuo for 24 hours. Thedried resin was subjected to HF as follows: to the dry peptide resin(0.4 g) in the HF reaction flask, anisole (2 mL) was added and thepeptide treated with 20 mL liquid HF at -20° C. for 2 hours. After theevaporation of the HF under vacuum, the anisole was washed with ether(20 mL, 5 times) and the solid residue dried in vacuo. The peptide wasextracted from the resin with TFA (10 mL 3 times) and the TFA evaporatedunder vacuum. The residue was dissolved in 20 mL 30% AcOH andlyophilized. This process was repeated 3 times. The crude peptide waspurified by semipreparative HPLC. The final product was obtained aswhite powder by lyophilization from dioxane, which gave 59 mg (34%) ofthe title compound.

Table 5 follows at this

                  TABLE 5    ______________________________________    PROCEDURE FOR 0.1 mMOLE SCALE    STEP SOLVENT/  VOLUME   TIME  REPEAT    NO.  REAGENT   (ML)     (MIN) (XS)   COMMENT    ______________________________________    1    DCM       10       2     3    2    TFA/DCM   10       2     1      Deprotection         55%    3    TFA/DCM   10       20    1      Deprotection         55%    4    DCM       10       2     3    5    NMP       10       2     4    6    DIEA/NMP  10       5     2      Neutralization         10%    7    NMP       10       2     5    8    TBTU/NMP/ 10       150   3      Cyclization         DIEA    9    NMP       10       2     4      Picric acid test.                                         If less than                                         98 ± 2%                                         Coupling perform                                         Steps 10.    10   TBTU/NMP/ 10       20 h  3      Cyclization,         DIEA                            Check pH, adjust                                         to pH 8 with                                         DIEA.    11   NMP       10       2     4      Picric acid test.                                         If less than                                         98 ± 2%                                         coupling perform                                         Step 12.    12   TBTU/NMP/ 10       120   3      Cyclization,         DIEA                            50° C. Check pH,                                         adjust to pH 8                                         with DIEA.    13   NMP       10       2     6    14   Piper-    10       10    1      Deprotection         idine/         NMP 20%    15   Piper-    10       1         idine/         NMP 20%    16   NMP       10       2     6      Check for                                         positive nin.    17   DCM       10       2     4    ______________________________________

HPLC RT 33.62 minutes (91%)

TOF MS: 1278 (M⁺)

AAA in agreement with the title compound

Example 4

H-D-Arg-Arg-cyclo(N.sup.α (4-propanoyl)Gly-Hyp-Phe-N.sup.α(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg--OH

Title compound was synthesized according to Example 3 except that instage 1, Fmoc-N.sup.α (3-Boc-amino-propylene)-S-Phe (0.326) wassubstituted for Fmoc-N.sup.α (3-Boc amino propylene)Gly. A total of0.643 g Boc-L-Arg(Tos)-O-resin (0.39 meq/g, 0.250 mmole) was used andreagent quantities were adjusted accordingly. Cyclic peptide yield (fromhalf the total resin used) was 74 mg (42%) of the title compound.

SPECIFIC EXAMPLES OF BUILDING UNITS

The following specific examples of novel building units are provided forillustrative purposes not meant to be limiting. "Procedures" aredetailed stepwise descriptions of synthetic procedures according to themore general schemes. "Methods" are general descriptions of analysesused to determine the progress of the synthetic process. Severalcompounds used in series produce "EXAMPLES" of novel building units ofthe present invention.

Procedure 1

Synthesis of N-Boc alkylene diamines (BocNH(CH₂)_(n) NH₂) (knowncompounds).

To a solution of 0.5 mole alkylene diamine in 0.5 L CHCl₃ cooled in anice-water bath, was added dropwise, with stirring, a solution of 10.91 g(0.05 mole) Boc₂ O in 0.25 L CHCl₃ for 3 h. The reaction mixture wasstirred for 16 h. at room temperature and then washed with water (8×250mL). The organic phase was dried over Na₂ SO₄ and evaporated to drynessin vacuo.

Procedure 2

Synthesis of N-Boc, N-Bzl alkylene diamines (BocNH(CH₂)_(n) NH-Bzl).

To a solution of 0.05 mole of mono Boc alkylene diamine in 60 mL MeOHwas added 2.77 mL (0.02 mole) Et₃ N, 9.02 g (0.075 mole) MgSO₄, and 5.56mL (0.055 mole) of freshly distilled benzaldehyde. The reaction mixturewas stirred under room temperature for 1.5 h. Then 11.34 g (0.3 mole) ofNaBH₄ were added in small portions during 0.5 h with cooling to -5° C.The reaction mixture was then stirred for 1 h at -5° C. and for another1 h at 0° C. The reaction was stopped by addition of 200 mL water andthe product was extracted with EtOAc (3×200 mL). The combined EtOAcextracts were washed with water (4×100 mL). The organic phase wasextract with 0.5N HCl (4×100 mL) and the aqueous solution wasneutralized under cooling with 25 mL 25% NH₄ OH, extracted with CHCl₃(3×100 ml) and the combined extracts were washed with water (3×80 mL),dried over Na₂ SO₄ and evaporated to dryness in vacuo.

Procedure 3

Synthesis of (R) or (S) α-hydroxy acids (known compounds).

To a solution of 16.52 g (0.1 mole) (R) or (S) amino acid in 150 ml 1NH₂ SO₄ was added dropwise a solution of 10.35 g (0.15 mole) NaNO₂ in 100mL H₂ O during 0.5 h with stirring and cooling in an ice bath. Thereaction mixture was stirred 3 h. at 0° C. and additional 18 h at roomtemperature, then the (R)- or (S)- hydroxy acid was extracted with etherin a continuous ether extractor. The etheral solution was washed with 1NHCl (2×50 mL), H₂ O (3×80 mL), dried over Na₂ SO₄ and evaporated todryness. The product was triturated twice from ether:petrol-ether(40°-60° C.) (1:10). The precipitate was filtered, washed with 50 mLpetrol-ether and dried.

Procedure 4

Synthesis of (R) or (S) α-hydroxy acid methyl esters (known compounds).

To a suspension of 0.065 mole (R)- or (S)- hydroxy acid in 100 mL etherwas added under cooling in an ice bath 300 mL of an etheral solution ofCH₂ N₂ until stable yellow color of reaction mixture was obtained. Thenthe ether solution was washed with 5% KHCO₃ (3×100 mL) and H₂ O (2×80mL), dried over Na₂ SO₄ and evaporated to dryness. The product was driedin vacuo.

Procedure 5

Synthesis of triflate of (R) or (S) α-hydroxy acid methyl esters.

To a cooled solution of 2.67 ml (0.033 mole) pyridine in 20 mL dry DCMwas added 5.55 mL (0.033 mole) Trf₂ O at -20° C. (dry ice in EtOH bath),then after 5 min a solution of 0.03 mole (R) or (S) α-hydroxy acidmethyl ester in 20 mL dry DCM was added dropwise. The reaction mixturewas stirred at room temperature for 45 min, then was passed through ashort silica gel column (2 cm). The product was eluted with 400 mL ofpetrol-ether:methylene-chloride (1:1). The solvent was evaporated invacuo.

Procedure 6

Synthesis of (R) or (S) N.sup.α (Bzl)(N.sup.ω -Boc-amino alkylene) aminoacid methyl esters ((R) or (S) BocNH(CH₂)_(n) N(Bzl)CH(R)COOMe).

To a solution of 0.022 mole of N.sup.α -Boc, N.sup.ω -Bzl alkylenediamine in 20 mL of dry DCM was added 3.04 mL (0.022 mole) Et₃ N. Then asolution of 0.02 mole of (R) or (S) α-hydroxy acid methyl ester triflatein 25 mL dry DCM was added dropwise (0.5 h.) under cooling in anice-water bath. The reaction mixture was stirred at room temperature for18 h. Then 150 mL of CHCl₃ was added and the yellow solution was washedwith water (3×80 mL). The organic phase was dried over Na₂ SO₄ andadsorbed on silica-gel and dried in vacuo. The silica-gel was washed onfilter with 0.5 L of petrol-ether and with 0.5 L of 2% EA in PE. Thenthe product was eluted from silica with 0.5 L of mixturepetrol-ether:ethyl-acetate (4:1). The solvent was evaporated in vacuo.If the product was not clean it was further purified on a small columnof silica-gel (250 mL). The first impurities were eluted with 0.8 L ofhexane then the product was eluted with 1.5 L of mixture ofpetrol-ether: ethyl-acetate (4:1).

Procedure 7

Hydrolysis of methyl esters

To a solution of 0.015 mole of methyl ester in 40 mL MeOH was added 10mL 7.5N NaOH cooled in an ice-water bath. The reaction mixture wasstirred at room temperature for approximately 24 h (until the methylester spot disappears on TLC). Then 100 mL of water were added and thereaction mixture was washed with petrol-ether (3×80 mL). The aqueoussolution was acidified under cooling by addition of 40 mL 2N HCL. Theproduct was extracted with a mixture of CHCl₃ :i-PrOH (3:1) (3×80 mL),dried over Na₂ SO₄, evaporated to dryness and dried in vacuo to obtain awhite foam in quantitative yield.

Procedure 8

Removal of Bzl by Hydrogenation with Pd/C

To a solution of 0.012 mole (R) or (S) N.sup.α (Bzl) (N.sup.ω -Boc-aminoalkylene) amino acid in 60 mL MeOH-DMF (11-1) was added 0.5 g 10% Pd/C.The solution was hydrogenated for 4 h under a pressure of 45-50 Psi atroom temperature. Then 200 mL of a mixture of DMF:MeOH:H₂ O:glacial AcOH(1:3:5:1) was added. The catalyst was filtrated off and washed (is theacetic acid?) with H₂ O or MeOH (2×15 mL). The combined filtrate wasevaporated to dryness and recrystallized from methanol: ether (15mL:250). The precipitate was filtered and dried in vacuo.

Procedure 9

Synthesis of (R) or (S) N.sup.α (Fmoc)(N.sup.ω -Boc-amino alkylene)amino acid.

To 50 mL water was added 0.07 mole of (R) or (S) N.sup.α (N.sup.ω-Boc-amino alkylene) amino acid and 1.95 mL (0.014 mole) Et₃ N. Thesuspension was stirred 2-3 h until a clear solution was obtained. Then asolution of 2.25 g (0.07 mole) of FmocOSu in 100 mL ACN was added. Thereaction mixture was stirred 18 h at room temperature, then 150 mL waterwas added and the solution was washed with petrol-ether (3×100 mL) andwith ether:petrol-ether (1:4). The aqueous solution was acidified byaddition of 14 mL 1N HCL. The product was extracted with EtOAc (4×100mL) and the organic phase was washed with 0.5N HCl (2×50 mL), H₂ O (3×80mL), dried over Na₂ SO₄, evaporated to dryness and recrystallized fromether: petrol-ether (80 mL: 200 mL)

Procedure 10

Synthesis of S-benzylcysteamine (Bzl-S-(CH₂)₂ -NH₂) (known compound).

To a suspension of 0.1 mole cysteamine hydrochloride in 20 mL methanolwere added 13.6 mL of 25% ammonia solution, followed by dropwiseaddition of 0.12 mole benzyl bromide at room temperature. The mixturewas stirred for 0.5 h, and the formed precipitate ofS-dibenzylcysteamine was collected by filtration. The product wasextracted with ether (3×100 mL) and the organic phase was successivelywashed with brine (2×100 mL), dried over MgSO₄ and the solventevaporated in vacuo. The crude product was essentially pure enough forthe next step. It could, however be recrystallized from ethyl acetate.Yield 86%, of white solid. m.p. 85°-6° C. NMR (CDCl₃) in agreement withthe title compound.

Example 5

N-(2-(benzylthio)ethylene)(S)leucine methyl ester

The title compound was prepared according to procedure 13 from theTriflate of (R) leucine methyl ester (Procedure 5).

Yield 70% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. Elemental analysis-calculated: C-65.05, H-8.53, N-4.74; found:C-66.29, H-9.03, N-4.49. (a)_(D14) =-51.2° (C 0.94, DCM).

Example 6

COMPOUND A:

N-(3-(benzylthio)propylene)(S)leucine methyl ester

The title compound was prepared from the Triflate of (R)leucine methylester (Procedure 5).

Yield 60% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. Elemental analysis-calculated: C-65.98, H-8.79, N-4.53; found:C-67.09, H-9.20, N-4.54. (a)_(D23) =-17.4° (C 1.44, DCM).

COMPOUND B:

N-(2-(benzylthio)ethylene)(S)phenylalanine methyl ester

The title compound was prepared from the Triflate of (R)phenyl lacticacid methyl ester (Procedure 5).

Yield 82% of white crystals. m.p.=48°-49° C. NMR (CDCl₃) in agreementwith the title compound. Elemental analysis-calculated: C-69.27, H-7.04,N-4.25; found: C-69.55, H-7.21, N-4.08. (a)_(D14) =-23.3° (C=1.01, DCM).

COMPOUND C:

N-(3-(benzylthio)propylene)(S)phenylalanine methyl ester

The title compound was prepared from the Triflate of (R)phenyl lacticacid methyl ester (Procedure 5).

Yield 71% of white crystals. m.p.=38°-39° C. NMR (CDCl₃) in agreementwith the title compound. Elemental analysis-calculated: C-69.94, H-7.34,N-4.08; found: C-69.66, H-7.39, N-4.37. (a)_(D26) =+2.0° (C 1.00, DCM).

COMPOUND D:

N-(4-(benzylthio)butylene)(S)phenylalanine methyl ester

The title compound was prepared from the Triflate of (S)phenyl lacticacid methyl ester (Procedure 5).

Yield 81% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. Elemental analysis-calculated: C-70.55, H-7.61, N-3.92; found:C-70.51, H-7.69, N-4.22. (a)_(D26) =+4.9° (C 1.00, DCM).

Example 7

COMPOUND E:

Boc-N-(2-(benzylthio)ethylene)glycine

The title compound was prepared.

Yield 88% of white crystals. m.p.=71°-72° C. NMR (CDCl₃) in agreementwith the title compound. Elemental analysis-calculated: C-59.05, H-7.12,N-4.30; found: C-59.39, H-7.26, N-4.18.

Example 8

COMPOUND F:

Boc-N-(2-(benzylthio)ethylene)(S)phenylalanine

The title compound was prepared from Compound B by hydrolysis.

Yield 78% of white crystals. m.p.=82°-83° C. NMR (CDCl₃) in agreementwith the title compound. (a)_(D25) =-105.9° (C 1.01, DCM).

Example 9

COMPOUND G:

Boc-N-(3-(benzylthio)propylene)(S)phenylalanine

The title compound was prepared from Compound C by hydrolysis.

Yield 99% of white crystals. m.p.=63°-64° C. NMR (CDCl₃) in agreementwith the title compound. (a)_(D25) =-87.4° (C 1.01, DCM).

Example 10

COMPOUND H:

Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)glycine ethyl ester

Boc-L-Phe was coupled to N-(2-(benzylthio)-ethylene)glycine ethyl ester.

Yield 32% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. Elemental analysis-calculated: C-64.77, H-7.25, N-5.60; found:C-64.39, H-7.02, N-5.53. (a)_(D16) =+4.5° (C 0.88, DCM).

Example 11

COMPOUND I:

Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)(S)phenylalanine methylester

Boc-L-Phe was coupled to N-(2-(benzylthio)ethylene) (S)phenylalaninemethyl ester (Compound B).

Yield 46% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. (a)_(D26) =-115.9° (C 1.0, CHCl₃).

COMPOUND J:

N-Bzl-β-alanine t-butyl ester

A solution of 6.16 g of β-alanine t-butyl ester acetate in 150 mL waterwas reacted with benzaldhyde (Procedure 2) to give 4.5 g, 64.5% yield

TLC Rf=0.78 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND K

N-Bzl-γ-amino butyric acid t-butyl ester

A solution of 6.58 g of γ-aminobutyric acid t-butyl ester acetate in 150mL water was reacted with benzaldhyde (Procedure 2) to give 4.24 g,57.9% yield

TLC Rf=0.74 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND L

N.sup.α (Bzl)(2-t-butyl carboxy ethylene)glycine benzyl ester

A solution of 3.53 g of N-Bzl-β-alanine t-butyl ester (Compound J) inDMF was reacted with 2.61 mL benzyl bromoacetate. Yield 86.9%

TLC Rf=0.95 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND M

N.sup.α (Bzl)(3-t-butyl carboxy propylene)glycine benzyl ester

A solution of 3.53 g of N-Bzl-γ-aminobutyric acid t-butyl ester(Compound K) in DMF was reacted with 2.61 mL benzyl bromoacetate. Yield83%

TLC Rf=0.92 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND N

N.sup.α (2-t-butyl carboxy ethylene)glycine

A solution of N.sup.α (Bzl)(2-t-butyl carboxy ethylene)glycine benzylester (Compound L) in MeOH was hydrogenated (Procedure 8). Yield 87.8%

TLC Rf=0.56 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND O

N.sup.α (3-t-butyl carboxy propylene)glycine

A solution of N.sup.α (Bzl) (3-t-butyl carboxy propylene) glycine benzylester (Compound M) in MeOH was hydrogenated (Procedure 8). Yield 94%

TLC Rf=0.3 (one spot); NMR (CDCl₃) in agreement with the title compound.

Example 12

COMPOUND P

N.sup.α (Fmoc)(2-t-butyl carboxy ethylene)glycine

A solution of N.sup.α (2-t-butyl carboxy ethylene)glycine (Compound N)in H₂ O:Et₃ N was reacted with FmocOSu (Procedure 9). Yield 90%

TLC Rf=0.5 (one spot); NMR (CDCl₃) in agreement with the title compound.

    ______________________________________    Elemental Analysis:                 % C         % H    % N    ______________________________________    Found:       67.38       6.34   3.11    Calc:        67.75       6.40   3.29    ______________________________________

Example 13

COMPOUND Q:

N.sup.α (Fmoc)(3-t-butyl carboxy propylene)glycine

A solution of N.sup.α (3-t-butyl carboxy propylene)glycine (Compound O)in H₂ O:Et₃ N was reacted with FmocOSu (Procedure 9). Yield 82%

TLC Rf=0.58 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

    ______________________________________    Elemental Analysis:                 % C         % H    % N    ______________________________________    Found:       68.29       6.83   3.88    Calc:        68.32       6.65   3.19    ______________________________________

COMPOUND R

(R)-O-Trf-3-Phenyllactic acid benzyl ester

To a cooled solution of Trf₂ O and pyridine in dry DCM (Procedure 5), asolution of 5.3 g of (R)-3-Phenyllactic acid benzyl ester was added.After the workup (Procedure 5), the yield was 91.43%. The product wasused immediately or kept in a cold desiccator under Ar.

COMPOUND S

N.sup.α (Bzl)(2-t-butyl carboxy ethylene)(S) Phenylalanine benzyl ester

A solution of 5.48 g of N-Bzl-β-alanine t-butyl ester (Compound 56) inDCM was reacted with 7.35 g of (R)-O-Trf-3-Phenyllactic acid benzylester (COMPOUND R) in dry DCM (Procedure 6). After workup the crudeproduct was purified by flash chromatography. PE:EtOAc (4:1) 1.5 L.After solvent evaporation under vacuum, the product was dried undervacuum.

Yield 71.5%; TLC Rf=0.77 (one spot); (α)_(D) =-62.7 (C=1, MeOH); NMR(CDCl₃) in agreement with the title compound.

COMPOUND T

N.sup.α (2-t-butyl carboxy ethylene)(S) Phenylalanine

A solution of 6.3 g of N.sup.α (Bzl)(2-t-butyl carboxy ethylene)(S)Phenylalanine benzyl ester (Compound S) in MeOH was hydrogenated(Procedure 8). Yield 48.6%

TLC Rf=0.52-0.54 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

Example 14

COMPOUND U:

N.sup.α (Fmoc)(2-t-butyl carboxy ethylene)(S) Phenylalanine

A solution of 2.13 g of N.sup.α (2-t-butyl carboxy ethylene)(S)Phenylalanine (Compound T) in H₂ O:Et₃ N was reacted with FmocOSu(Procedure 9). Yield 38%

TLC Rf=0.77 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

    ______________________________________    Elemental Analysis:                 % C         % H    % N    ______________________________________    Found:       71.92       639    2.87    Calc:        72.21       6.45   2.72    ______________________________________     HPLC 93%

COMPOUND V

N.sup.α (Bzl)(2-Boc amino ethylene)glycine benzyl ester

A solution of 0.0325 mole of N-Boc, N-Bzl,1,2 diaminoethane (Compound 5)in DMF was reacted with 5.15 mL benzyl bromoacetate. Yield 97.9%

TLC Rf=0.78 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND W

N.sup.α (Bzl)(3-Boc amino propylene)glycine benzyl ester

A solution of 0.0325 mole of N-Boc, N-Bzl,1,3 diaminopropane in DMF wasreacted with 5.15 mL benzyl bromoacetate. Yield 98.2%

TLC Rf=0.78 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND X

N.sup.α (Bzl)(4-Boc amino butylene)glycine benzyl ester

A solution of 0.0325 mole of N-Boc, N-Bzl,1,4 diaminobutane in DMF wasreacted with 5.15 mL benzyl bromoacetate. Yield 98.8%

TLC Rf=0.82 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND Y

N.sup.α (Bzl)(6-Boc amino hexylene)glycine benzyl ester

A solution of 0.0325 mole of N-Boc, N-Bzl,1,6 diaminohexane in DMF wasreacted with 5.15 mL benzyl bromoacetate. Yield 98.8%

TLC Rf=0.79 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND Z

N.sup.α (2-Boc amino ethylene)glycine

A solution of 0.025 mole of N.sup.α (Bzl)(2-Boc amino ethylene)glycinebenzyl ester (Compound V) in 60 mL MeOH was hydrogenated (Procedure 8).Yield 85% of white solid. mp 200°-2° C.

TLC Rf=0.22 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND AA

N.sup.α (3-Boc amino propylene)glycine

A solution of 0.025 mole of N.sup.α (Bzl) (3-Boc amino propylene)glycinebenzyl ester (Compound W) in 60 mL MeOH was hydrogenated (Procedure 8).Yield 74% of white solid. mp 214°-6° C.

TLC Rf=0.27 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND AB

N.sup.α (4-Boc amino butylene)glycine

A solution of 0.025 mole of N.sup.α (Bzl) (4-Boc amino butylene)glycinebenzyl ester (Compound X) in 60 mL MeOH was hydrogenated (Procedure 8).Yield 89.5% of white solid. mp 176°-8° C.

TLC Rf=0.23 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND AC

N.sup.α (6-Boc amino hexylene)glycine

A solution of 0.025 mole of N.sup.α (Bzl) (6-Boc amino hexylene)glycinebenzyl ester (Compound Y) in 60 mL MeOH was hydrogenated (Procedure 8).Yield 80% of white solid. mp 172°-4° C.

TLC Rf=0.26 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

Example 15

COMPOUND AD:

N.sup.α (Fmoc) (2-Boc amino ethylene)glycine

A solution of 0.02 mole of N.sup.α (2-Boc amino ethylene)glycine(Compound Z) in H₂ O:Et₃ N was reacted with FmocOSu (Procedure 9). Yield80% of white solid.

mp 130°-132° C. TLC Rf=0.5 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

    ______________________________________    Elemental Analysis:                 % C         % H    % N    ______________________________________    Found:       65.18       6.11   5.91    Calc:        65.43       6.40   6.63    ______________________________________

Example 16

COMPOUND AE:

N.sup.α (Fmoc)(3-Boc amino propylene)glycine

A solution of 0.02 mole of N.sup.α (Fmoc)(3-Boc amino propylene)glycine(Compound AA) in H₂ O:Et₃ N was reacted with FmocOSu (Procedure 9).Yield 85% of white solid. mp 125° C.

TLC Rf=0.5-0.6 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

    ______________________________________    Elemental Analysis:                 % C         % H    % N    ______________________________________    Found:       66.05       6.65   6.00    Calc:        66.06       6.65   6.16    ______________________________________

Example 17

COMPOUND AF:

N.sup.α (Fmoc)(4-Boc amino butylene)glycine

A solution of 0.02 mole of N.sup.α (Fmoc) (4-Boc amino butylene)glycine(Compound AB) in H₂ O:Et₃ N was reacted with FmocOSu (Procedure 9).Yield 79.4% of white solid. mp 150°-152° C.

TLC Rf=0.42-0.47 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

    ______________________________________    Elemental Analysis:                 % C         % H    % N    ______________________________________    Found:       66.35       6.84   5.77    Calc:        66.06       6.88   5.98    ______________________________________

Example 18

COMPOUND AG:

N.sup.α (Fmoc)(6-Boc amino hexylene)glycine

A solution of 0.02 mole of N.sup.α (Fmoc) (6-Boc amino hexylene)glycine(Compound AC) in H₂ O:Et₃ N was reacted with FmocOSu (Procedure 9).Yield 81.5% of white solid. mp 78°-80° C. TLC Rf=0.7 (one spot);

NMR (CDCl₃) in agreement with the title compound.

    ______________________________________    Elemental Analysis:                 % C         % H    % N    ______________________________________    Found:       68.02       7.08   5.37    Calc:        67.72       7.31   5.67    ______________________________________

SYNTHETIC EXAMPLES

Two series of octapeptide somatostatin analogs of the present inventionwere synthesized, characterized, and tested for biological activity.

1) The first series of compounds corresponds to the general Formula(XIVb); this series comprises compounds of the specific formula

    H-(D)Phe-R.sup.6 -Phe-(D)Trp-Lys-Thr-R.sup.11 -Thr-NH.sub.2

wherein R⁶ and R¹¹ are N.sup.α ω-functionalized alkylene amino acidbuilding units.

2) The second series of compounds corresponds to the general Formula(XVIc); this series comprises compounds of the specific formula

    H-(D)Phe-R.sup.6 -Phe-(D)Trp-Lys-R.sup.10 -Thr-NH.sub.2

wherein R⁶ and R¹⁰ are N.sup.α ω-functionalized alkylene amino acidbuilding units.

The structures of these novel synthetic peptide analogs into whichN.sup.α ω-functionalized amino acid building units were incorporated,are summarized in Tables 7 and 8. In both series, the building unitsused were glycine building units in which the bridging groups, attachedvia the alpha nitrogens to the peptide backbone, were varied.

For the sake of simplicity, these two series are referred to herein asthe SST Gly⁶,Gly¹¹ and SST Gly⁶,Gly¹⁰ series, respectively.

In each series, the position of the cyclization points was constant,while the length and direction of the bridge was varied. Thus, C2,N2refers to a bridge consisting of an amide bond in which the carbonylgroup is closer to the amino end of the peptide and which contains twomethylene groups between the bridge amide and each of the backbonenitrogens involved in the bridge.

Peptide assembly was carried out either manually or with an automaticpeptide synthesizer (Applied Biosystems Model 433A). Following peptideassembly, de-protection of bridging groups that form the cyclizationarms was carried out with Pd(PPh₃)₄ (palladium tetrakis triphenylphosphine) in the case of Allyl/Alloc protecting groups or with TFA inthe case of tBu/Boc protecting groups. For obtaining the linear(non-cyclized) analog, the peptides were cleaved from the resin at thisstage. Cyclization of the peptides was carried out with PyBOP. Cleavageof the peptides from the polymeric support was carried out with suitablereagents depending on the type of resin used, e.g., with TFA for Rinkamide type resins and with HF for mBHA (para-methyl benzhydryl amine)type resins. The crude products were characterized by analytical HPLC.The peptides were purified by preparative reversed phase HPLC. Thepurified products where characterized by analytical HPLC, massspectroscopy, and amino acid analysis.

                  TABLE 7    ______________________________________    SST Gly.sup.6,Gly.sup.11    Example Bridging    Compound          Crude    No.     Groups      Number     Method Yield    ______________________________________    16      C1,N2 Cyclic                        DE-3-32-4  1      NA**    17      C1,N2 Linear                        DE-3-32-2  1      NA    18      C1,N3 Cyclic                        PTR 3004   2      79 mg    19      C1,N3 Linear                        PTR 3005   2      34 mg    20      C2,N2 Cyclic                        PTR 3002   1      NA    21      C2,N2 Linear                        PTR 3001   1      NA    22      C2,N3 Cyclic                        PTR 3007   2      40 mg    23      C2,N3 Linear                        PTR 3008   2      40 mg    24      N2,C2 Cyclic                        YD-9-166-1 2      NA    25      N2,C2 Linear                        YD-9-168-1 2      NA    26      N3,C2 Cyclic                        PTR 3010   2      100 mg    27      N3,C2 Linear                        PTR 3011   2      NA    28      Linear*     PTR 3003   3      96 mg    ______________________________________     *Linear refers to the identical sequence with Gly residues in place of     R.sup.6 and R.sup.11.     **NA denotes not available.     Table 7 methods:     1) Manual synthesis on mBHA resin. HF cleavage.     2) Manual synthesis on Rapp tentagel resin. TFA cleavage.     3) Rink amide resin; assembly in automated peptide synthesizer, 0.1 mmol     scale.

                  TABLE 8    ______________________________________    SST Gly.sup.6,Gly.sup.10    Example Bridging    Compound          Crude    No.     Groups      Number     Method Yield    ______________________________________    29      C1,N2 Cyclic                        YD-9-171-3 1      20   mg    30      C1,N2 Linear                        YD-9-171-2 1      10   mg    31      C1,N3 Cyclic                        YD-9-175-3 1      44.9 mg    32      C1,N3 Linear                        YD-9-175-2 1      25.4 mg    33      C2,N2 Cyclic                        PTR 3019   1      40   mg    34      C2,N2 Linear                        PTR 3020   1      26   mg    35      C2,N3 Cyclic                        YD-5-28-3  3      101.5                                               mg    36      C2,N3 Linear                        YD-5-28-2  3      48.3 mg    37      N2,C2 Cyclic                        PTR 3016   2      60   mg    38      N2,C2 Linear                        PTR 3017   2      40   mg    39      N3,C2 Cyclic                        YS-8-153-1 2      93   mg    40      N3,C2 Linear                        YS-8-152-1 2      54   mg    41      *Linear     PTR 3021   1      100  mg            **Acetylated            Des-D-Phe.sup.5    52      N3,C2 Cyclic                        PTR 3013          67   mg    53      N3,C2 Linear                        PTR 3014          48   mg    ______________________________________     *Linear refers to the identical sequence with Gly residues in place of     R.sup.6 and R.sup.10.     **Acetylated DesD-Phe.sup.5 refers to the same sequence in which the N     terminal DPhe.sup.5 is absent and the Nterminus is acetylated.     Table 8 methods:     1) Assembly in automated peptide synthesizer; 0.1 mmol scale. (HBTU).     2) Manual synthesis; PyBrop.     3) Assembly in automated peptide synthesizer, 0.25 mmol scale. (HBTU).

Synthesis of SST Gly⁶,Gly¹⁰ N3,C2:

Five grams of Rink amide resin (NOVA) (0.49 mmol/g), were swelled inN-methylpyrrolidone (NMP) in a reaction vessel equipped with a sinteredglass bottom and placed on a shaker. The Fmoc protecting group wasremoved from the resin by reaction with 20% piperidine in NMP (2 times10 minutes, 25 ml each). Fmoc removal was monitored by ultravioletabsorption measurement at 290 nm. A coupling cycle was carried out withFmoc-Thr(OtBu)--OH (3 equivalents) PyBrop (3 equivalents) DIEA (6equivalents) in NMP (20 ml) for 2 hours at room temperature. Reactioncompletion was monitored by the qualitative ninhydrin test (Kaisertest). Following coupling, the peptide-resin was washed with NMP (7times with 25 ml NMP, 2 minutes each). Capping was carried out byreaction of the peptide-resin with acetic anhydride (capping mixture:HOBt 400 mg, NMP 20 ml, acetic anhydride 10 ml, DIEA 4.4 ml) for 0.5hours at room temperature. After capping, NMP washes were carried out asabove (7 times, 2 minutes each). Fmoc removal was carried out as above.Fmoc-Phe--OH was coupled in the same manner, and the Fmoc group removed,as above. The peptide resin was reacted with Fmoc-Gly-C2 (Allyl)building unit: coupling conditions were as above. Fmoc removal wascarried out as above. Fmoc-Lys(Boc)--OH was coupled to the peptide resinby reaction with HATU (3 equivalents) and DIEA (6 equivalents) at roomtemperature overnight and then at 50° C. for one hour. Additional DIEAwas added during reaction to maintain a basic medium (as determined bypH paper to be about 9). This coupling was repeated. Coupling completionwas monitored by the Fmoc test (a sample of the peptide resin was takenand weighed, the Fmoc was removed as above, and the ultravioletabsorption was measured). Fmoc-D-Trp--OH was coupled to the peptideresin with PyBrop, as described above. Following Fmoc removal,Fmoc-Phe--OH was coupled in the same way. Synthesis was continued withone-fifth of the peptide resin.

Following Fmoc removal, the second building unit was introduced:Fmoc-Gly-N3(Alloc)--OH by reaction with PYBrop, as described above.Capping was carried out as described above. Following Fmoc removal, thepeptide-resin was divided into two equal portions. Synthesis wascontinued with one of these portions. Boc-D-Phe--OH was coupled byreaction with HATU, as described above for Fmoc-Lys(Boc)--OH. Cappingwas carried out as above.

The Allyl and Alloc protecting groups were removed by reaction withPd(PPh₃)₄ and acetic acid 5%, morpholine 2.5% in chloroform, underargon, for 2 hours at room temperature. The peptide resin was washedwith NMP as above. Two-thirds of the resin were taken for cyclization.Cyclization was carried out with PyBOP 3 equivalents, DIEA 6equivalents, in NMP, at room temperature overnight. The peptide resinwas washed and dried. The peptide was cleaved from the resin by reactionwith TFA 81.5%, phenol 5%, water 5%, EDT 2.5%, TIS(tri-isopropyl-silane) 1%, and 5% methylene chloride, at 0° C. for 15minutes and 2 hours at room temperature under argon. The mixture wasfiltered into cold ether (30 ml, 0° C.) and the resin was washed with asmall volume of TFA. The filtrate was placed in a rotary evaporator andall the volatile components were removed. An oily product was obtained.It was triturated with ether and the ether decanted, three times. Awhite powder was obtained. This crude product was dried. The weight ofthe crude product was 93 mg.

PHYSIOLOGICAL EXAMPLES Example 44

BRADYKININ ANTAGONIST ASSAY (Displacement of (³ H)dopamine release fromPC 12 cells)

Novel backbone cyclized peptide analogs of the present invention wereassayed in vitro for bradykinin antagonist activity by protection of (³H)dopamine release from PC 12 cells that express bradykinin receptors.PC12 cells were grown in Dulbecco Modified Eagle's medium with highglucose, supplemented with 10% horse serum, 5% fetal calf serum, 130units/ml penicillin and 0.1 mg/ml streptomycin. For experiments, cellswere removed from the medium using 1 mmole EDTA and replated on collagencoated-12- well plates and assayed 24 hr later. Release of (³ H)dopaminewas determined as follows: cells were incubated for 1.5 hr at 37° C.with 0.5 ml of growth medium and 0.85 ml (³ H)DA (41 Ci/mmole) and 10mg/ml pargyline followed by extensive washing with medium (3×1 ml) andrelease buffer consisting of (mM): 130 NaCl; 5 KCl; 25 NaHCO₃ ; 1 NaH₂PO₄ ; 10 glucose and 1.8 CaCl₂. In a typical experiment, cells wereincubated with 0.5 ml buffer for 5 consecutive incubation periods of 3min each at 37° C. Spontaneous (³ H)DA release was measured bycollecting the medium released by the cells successively for the first 3min period. Antagonists were added to the cells 3 min prior tostimulation (at the second period), and stimulation of (³ H)DA releaseby 100 nmole of bradykinin are monitored during the 3 period by 60 mmoleKCl. The remaining of the (³ H)DA was extracted from the cells by overnight incubation with 0.5 ml 0.1N HCl. (³ H)DA release during each 3 minperiod was expressed as a % of the total (³ H)DA content of the cells.Net evoked release was calculated from (³ H)DA release duringstimulation period after subtracting basal (³ H)DA release in thepreceding baseline period if not indicated otherwise.

At 10⁻⁶ M, Example 1 showed 30% inhibition of BK activity, Example 4showed 17% inhibition of BK activity. Note, the noncyclized (control)peptide of Example 2 showed 0% inhibition of BK activity.

Example 45

BRADYKININ ANTAGONIST ASSAY (Guinea-pig assay)

The ileum of the guinea-pig was selected as the preparation for thebioassay. This tissue contains predominantly BK₂ receptors. Thepreparation consists of the longitudinal muscle layer with the adheringmesenteric plexus. The isolated preparation was kept in Krebs solutionand contractions were measured with an isometric force transducer. Theguinea-pig ileum is highly sensitive to BK, with EC₅₀ at 2×10⁻⁸ M. Atleast two control responses to BK (2×10⁻⁸ M) were measured previous tomeasuring the responses of backbone cyclized peptides of the presentinvention. Atropine (1 μM) was always present.

At 10⁻⁶ M, Example 1 showed 24% inhibition of BK activity, Example 3showed 10% and Example 4 showed 17% inhibition of BK activity. Note, thenoncyclized (control) peptide of example 2 showed 0% inhibition of BKactivity.

Example 46

SOMATOSTATIN ASSAY (Receptor based screening)

Initial screening is conducted using ¹²⁵ I-labeled SST analogs andpituitary membrane preparations or cell lines. The binding assay isdescribed in Tran, V. T., Beal, M. F. and Martin, J. B. Science,228:294-495, 1985, which is incorporated herein by reference in itsentirety and is optimized with regards to membrane concentration,temperature and time. The assay is sensitive (nM range) and robust.Selectivity will be based on the recent cloning of the five human SSTreceptors. The ability to screen the compounds with regard to bindingand biological activity in mammalian cells should facilitate thedevelopment of subtype-selective analogs. These compounds are useful inthe treatment of specific endocrine disorders and therefore should bedevoid of unwanted side effects.

Example 47

SOMATOSTATIN (SST) ASSAY (In vivo assays)

The biological effects of SST on growth hormone, insulin and glucagonrelease is conducted by measuring the levels of these hormones usingcommercially available RIA test kits. Pharmacological effects of SST inpatients with neuroendocrine tumors of the gut will requiredetermination of 5-hydroxyindole acetic acid (for carcinoid) and VIP(for VIPoma). In vivo visualization of SST receptor-positive tumors isperformed as described by Lambert et al., New England J. Med.,323:1246-1249 1990, following i.v. administration of radio-iodinated SSTanalogs.

Example 48

Receptor binding specificity of cyclic peptide analogs

Binding of representative peptides of Examples 39-54 to differentsomatostatin receptors was measured in vitro, in Chinese Hamster Ovary(CHO) cells expressing the various receptors. An example of theselectivity obtained with the cyclic peptides is presented in Table 9.The values presented are percent inhibition of radioactive iodinatedsomatostatin (SRIF-14) binding.

                                      TABLE 9    __________________________________________________________________________    Binding of peptide analogs to somatostatin receptor subtypes    Conc. (M)    Somatostatin Receptor (SSTR) Subtype    Compound          Compound                 SSTR.sub.--                     2B      SSTR.sub.--                                 5    Number          Description                 10.sup.-6                     10.sup.-7                         10.sup.-8                             10.sup.-6                                 10.sup.-7                                     10.sup.-8    __________________________________________________________________________    PTR 3003          Linear 16  3   0   55  20  0    PTR 3004          Cyclic C1, N3                 0   0   0   14  0   0    PTR 3005          Linear C1, N3                 0   0   0    9  0   0    PTR 3007          Cyclic C2, N3                 0   0   0   19  9   0    PTR 3008          Linear C2, N3                 0   0   0   15  6   0    PTR 3010          Cyclic N3, C2                 0   0   0   63  26  9    PTR 3011          Cyclic N3, C2                 0   0   0   27  66  27    Control    Peptides    BIM 3503          Pos. Control                 81  33  16  92  66  27    PTR 4003          Neg. Control                 0   0   0   0   0   0    __________________________________________________________________________

Example 49

Resistance to biodegradation of SST analogs

The in vitro biostability of a SST cyclic peptide analog, PTR 3002, wasmeasured in human serum, and was compared to the same sequence in anon-cyclic peptide analog (PTR 3001), to octreotide (Sandostatin), andto native somatostatin (SRIF). The results are shown in FIG. 1. In thisassay, the cyclic peptide in accordance with the present invention is asstable as octreotide, is more stable than the corresponding non-cyclicstructure, and is much more stable than SRIF. The assay was based onHPLC determination of peptide degradation as a function of time at 37°C.

Example 50

Inhibition of growth hormone release by SST analogs

In vivo determination of the pharmacodynamic properties of cyclicpeptide analogs was carried out. Inhibition of Growth Hormone (GH)release as a result of peptide administration was measured. Measurementswere carried out in Sprague-Dawley male rats: peptide analog activitywas compared in this study to SRIF or to octreotide (Sandostatin). Eachgroup consisted of 4 rats. Time course profiles for GH release underconstant experimental conditions were measured.

Methods

Adult male Sprague-Dawley rats, specific pathogen free (SPF), weighing200-350 g, were maintained on a constant light-dark cycle (light from8:00 to 20:00 h), temperature (21±3° C.), and relative humidity(55±10%). Laboratory chow and tap water were available ad libitum. Onthe day of the experiment, rats were anesthetized with pentobarbitone(50 mg/kg). Rats anesthetized with pentobarbitone exhibit lowsomatostatin levels in portal blood vessels. (Plotsky, P. M., Science,230, 461-463, 1985). A single blood sample (0.6 ml) was taken from theexposed cannulated jugular vein for the determination of the basal GHlevels (-15 min). Immediately thereafter the appropriate peptidepretreatment was administered. The animals received 10 μg/kg of eithernative somatostatin (SRIF) or the synthetic analog octreotide(Sandostatin), or the cyclic peptide analog. A saline solution (0.9%NaCl) was administered as a control. All peptides were administeredsubcutaneously in a final volume of 0.2 ml. Further sampling was carriedout at 15, 30, 60, and 90 minutes after peptide administration.Immediately after the collection of each blood sample, an appropriatevolume (0.6 ml) of saline was administered intravenously. Blood sampleswere collected into tubes containing heparin (15 unites per ml of blood)and centrifuged immediately. Plasma was separated and kept frozen at-20° C. until assayed.

Rat growth hormone (rGH) ¹²⁵ I! levels were determined by appropriateradioimmunoassay kit (Amersham). The standard in this kit has beencalibrated against a reference standard preparation (NIH-RP2) obtainedfrom the National Institute of Diabetes and Digestive and KidneyDiseases. All samples were measured in duplicate.

Example 51

Lack of toxicity of cyclized peptide analogs

PTR 3007 at a dose of 1.5 mg/kg was well tolerated after singleintraperitoneal application. PTR 3013 was not toxic to the rats evenwith doses of 4 mg/kg. These two doses are several orders of magnitudehigher than those needed to elicit the desired endocrine effect. Thepeptides dissolved in saline produced no untoward side effects on thecentral nervous system, cardiovascular system, body temperature, nor onthe periphery of the animals. Rats were observed for 4 hours postadministration of the peptides. PTR 3007 and 3013 produced norespiratory disturbances, did not result in the appearance ofstereotyped behavior, or produce any changes in muscle tone. After 3hours, postmortem examination did not detect any abnormality in theliver, kidneys, arteries and veins, gastrointestinal tract, lungs,genital system, nor the spleen.

What is claimed is:
 1. An ω-functionalized amino acid derivative of thegeneral Formula: ##STR42## wherein X is an alkylene group; R is the sidechain of an amino acid;B is a protecting group selected from the groupconsisting of alkyloxy, substituted alkyloxy, and aryloxy carbonyls; Gis a functional group selected from the group consisting of amines,thiols, alcohols, carboxylic acids and esters, aldehydes and alkylhalides; and A is a protecting group thereof; wherein A and B aredifferent; with the proviso that if G is an amine, R is other thanhydrogen, methyl or iso-butyl.
 2. The ω-functionalized amino acidderivative of claim 1 wherein G is a thiol group.
 3. Theω-functionalized amino acid derivative of claim 1 wherein G is acarboxyl group.
 4. The ω-functionalized amino acid derivative of claim 1wherein R is benzyl, methyl, or isobutyl.
 5. The ω-functionalized aminoacid derivative of claim 1 wherein G is an amine group with the provisothat R is CH₃ --, (CH₃)₂ CH--, (CH₃)₂ CHCH₂ --, CH₃ CH₂ CH(CH₃)--, CH₃S(CH₂)₂ --, HOCH₂ --, CH₃ CH(OH)--, HSCH₂ --, NH₂ C (═O)CH₂ --, NH₂C(═O)(CH₂)₂ --, NH₂ (CH₂)₃ --, HOC(═O)CH₂ --, HOC(═O)(CH₂)₂ --, NH₂(CH₂)₄ --, C(NH₂)₂ NH(CH₂)₃ --, HO--phenyl--CH₂ --, benzyl,methylindole, or methylimidazole.
 6. The ω-functionalized amino acidderivative of claim 1 wherein R is protected with a specific protectinggroup.
 7. The ω-functionalized amino acid derivative of claim 1 whereinG is not an amine.
 8. The ω-functionalized amino acid derivative ofclaim 1 wherein the A and B protecting groups are orthogonal to eachother.
 9. The ω-functionalized amino acid derivative of claim 1 whereinR is the side chain of (S)- or (R)-alanine; (S)- or (R)-phenylalanine;glycine; or leucine.
 10. The ω-functionalized amino acid derivative ofclaim 1 wherein R is the side chain of an amino acid that is bound witha protecting group and the A and B protecting groups are orthogonal tothe protecting group of the amino acid.
 11. The ω-functionalized aminoacid derivative of claim 10 wherein R is the side chain of (S)- or(R)-alanine; or (S)- or (R)-phenylalanine.
 12. An ω-functionalized aminoacid derivative selected from the group consisting of:a) N.sup.α-(Fmoc)(3-Boc-amino propylene)-(S)Phenylalanine; b) N.sup.α-(Fmoc)(3-Boc-amino propylene)-(R)Phenylalanine; c) N.sup.α-(Fmoc)(4-Boc-amino butylene)-(S)Phenylalanine; d) N.sup.α-(2-(benzylthio)ethylene)glycine ethyl ester; e) N.sup.α-(2-(benzylthio)ethylene)(S)leucine methyl ester; f) N.sup.α-(3-(benzylthio)propylene)(S)leucine methyl ester; g) Boc-N.sup.α-(2-(benzylthio)ethylene) glycine; h) Boc-N.sup.α-(2-(benzylthio)ethylene)(S)phenylalanine; i) Boc-N.sup.α-(3-(benzylthio)propylene)(S)phenylalanine; j)Boc-L-phenylalanyl-N.sup.α -(2-(benzylthio)ethylene)glycine-ethyl ester;k) Boc-L-phenylalanyl-N.sup.α -(2-(benzylthio)ethylene)-(S)phenylalaninemethyl ester; l) N.sup.α (Fmoc)-(2-t-butyl carboxy ethylene)glycine; m)N.sup.α (Fmoc)-(3-t-butyl carboxy propylene)glycine; and n) N.sup.α(Fmoc)(2-t-butyl carboxy ethylene)(S) phenylalanine.
 13. A method ofmaking an ω-functionalized amino acid derivative of the general Formula:##STR43## wherein X is a spacer group selected from the group consistingof alkylene, substituted alkylene, arylene, cycloalkylene andsubstituted cycloalkylene; R is the side chain of an amino acid; A and Bare protecting groups selected from the group consisting of alkyloxy,substituted alkyloxy, or aryloxy carbonyls;said methodcomprising:reacting a diamine compound of the general Formula: ##STR44##wherein A, B and X are as defined above, with a triflate of Formula CF₃SO₂ --O--CH(R)--CO--E wherein E is a carboxyl protecting group and R isas defined above; to yield a compound of Formula: ##STR45## wherein A,B, E, R and X are as defined above and deprotecting the carboxyl toyield an N.sup.α ω-functionalized amino acid derivative, wherein theω-functional group is an amine.
 14. A method of making anω-functionalized amino acid derivative of the general Formula: ##STR46##wherein B is a protecting group selected from the group of alkyloxy,substituted alkyloxy, or aryloxy carbonyls; R is the side chain of anamino acid; X is a spacer group selected from the group of alkylene,substituted alkylene, arylene, cycloalkylene or substitutedcycloalkylene; and A is a protecting group selected from the group ofalkyl or substituted alkyl, thio ether or aryl or substituted aryl thioether;comprising the steps of:i) reacting a compound of the generalFormula B--NH--X--S--A with a triflate of the general Formula CF₃ SO₂--O--CH(R)--CO--E wherein E is a carboxyl protecting group and A, X andR are as defined above, to give a compound of the Formula: ##STR47## ii)selectively removing the protecting group E, and protecting the freeamino group to yield an N.sup.α (ω-functionalized) amino acidderivative, wherein the ω-functional group is a thiol.
 15. A method ofmaking an ω-functionalized amino acid derivative of the general Formula:##STR48## where B is a protecting group selected from the group ofalkyloxy, substituted alkyloxy, or aryloxy carbonyls; R is the sidechain of an amino acid; X is a spacer group selected from the group ofalkylene, substituted alkylene, arylene, cycloalkylene or substitutedcycloalkylene; and A is a protecting group selected from the group ofalkyl or substituted alkyl, esters, or thio esters or substituted arylesters or thio esters;comprising the steps of:i) reacting a compound ofthe general Formula B--NH--X--CO--A with a triflate of the generalFormula CF₃ SO₂ --O--CH(R)--CO--E wherein E is a carboxyl protectinggroup and A, B, X and R are as defined above, to give a compound ofFormula: ##STR49## and selectively removing protecting group E, to yieldan N.sup.α (ω-functionalized) amino acid derivative, wherein theω-functional group is a carboxyl.